Methods of administering tetrahydrobiopterin, associated compositions, and methods of measuring

ABSTRACT

The present invention is directed to treatment methods of administering tetrahydrobiopterin, including in oral dosage forms, in intravenous formulations, and with food. Also disclosed herein are biopterin assays for measuring the amount of biopterin and metabolites of biopterin in a sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/329,828 filed Dec. 8, 2008, which in turn is a continuation ofInternational Application No. PCT/US08/060,041, filed Apr. 11, 2008,which claims priority to U.S. Provisional Application Nos. 60/922,821,filed Apr. 11, 2007, and 61/019,753, filed Jan. 8, 2008, the disclosuresof which are incorporated herein by reference in their entirety.

BACKGROUND

1. Field

The present invention is generally directed to compositions and methodsfor treating BH4-responsive disorders, and methods and compositions fordetecting and quantitating biopterins.

2. Background of the Related Technology

Tetrahydrobiopterin (referred to herein as BH4) is a biogenic amine ofthe naturally-occurring pterin family that is a cofactor for a number ofdifferent enzymes, including phenylalanine hydroxylase (PAH), tyrosinehydroxylase, tryptophan hydroxylase and nitric oxide synthase. Pterinsare present in physiological fluids and tissues in reduced and oxidizedforms, however, only the 5, 6, 7, 8, tetrahydrobiopterin is biologicallyactive. It is a chiral molecule and the 6R enantiomer of the cofactor isknown to be the biologically active enantiomer. For a detailed review ofthe synthesis and disorders of BH4 see Blau et al., 2001 (Disorders oftetrahydrobiopterin and related biogenic amines. In: Scriver C R,Beaudet A L, Sly W S, Valle D, Childs B, Vogelstein B, eds. TheMetabolic and Molecular Bases of Inherited Disease. 8th ed. New York:McGraw-Hill, 2001: 1275-1776).

Fiege, et al., Molecular Genetics and Metabolism 81:45-51 (2004) studiedpharmacokinetics of orally administered tetrahydrobiopterin (BH4) andsuggested a “rather large variability of orally administered BH4,probably due to different absorption in the gut and/or to the firstpassage effect.”

Use of tetrahydrobiopterin has been proposed for treating a variety ofdifferent disease states, and there exists a need for alternative andimproved methods of administering this drug.

SUMMARY OF THE INVENTION

The present invention relates to methods of administering6R-(L-erythro)-5,6,7,8-tetrahydrobiopterin (BH4), or a pharmaceuticallyacceptable salt thereof, in a manner that improves or maximizes its oralbioavailability and/or improves or optimizes the consistency of oralbioavailability from one administration to the next. Such methods can beapplied in the treatment of any BH4-responsive disorder, includingmetabolic diseases, cardiovascular diseases, anemia, andneuropsychiatric disorders. The methods of the invention advantageouslyallow better control of clinical symptoms, e.g. reduced fluctuation inplasma phenylalanine levels, blood pressure, neurotransmitter levels, orother clinical parameters.

As used herein, BH4 refers to6R-(L-erythro)-5,6,7,8-tetrahydrobiopterin. The term BH4 as used hereinis also to be understood to optionally mean a pharmaceuticallyacceptable salt of 6R-(L-erythro)-5,6,7,8-tetrahydrobiopterin, unlessthe context dictates otherwise.

In a first aspect, the invention provides methods of orallyadministering to a patient in need thereof a purified preparation ofBH4.

In an exemplary embodiment, the methods comprise the step of informingthe patient that absorption of tetrahydrobiopterin is increased when itis ingested with food compared to when ingested without food. In someembodiments, the patient is informed that ingestion shortly following ameal, for example, a high-fat, high-calorie meal, results in an increasein any one, two, three or all of the following parameters: mean plasmaconcentration, Cmax, AUC, AUC(0-t) and/or AUC(inf). In exemplaryembodiments, the patient is informed that administration of BH4 with ahigh-fat meal increases Cmax and AUC compared to administration of BH4without food (in a fasting condition). In some embodiments, the relativeincrease can be at least 20% or 30% or more.

In alternative embodiments or in addition to the preceding embodiments,the method of administering tetrahydrobiopterin comprises informing thepatient that absorption of tetrahydrobiopterin is increased wheningested as an intact tablet compared to when ingested after beingdissolved in liquid. In some embodiments, the patient is informed thatingestion of intact tablets results in an increase in any of thefollowing parameters: mean plasma concentration, Cmax, AUC, AUC(0-t) orAUC(inf). In exemplary embodiments, the patient is informed thatadministration of BH4 as an intact tablet increases Cmax and AUCcompared to administration of BH4 after being dissolved in a liquid. Insome embodiments, the relative increase can be at least 20% or more.

Any of the preceding methods may be carried out by providing oradministering tetrahydrobiopterin in a container containing printedlabeling informing the patient of the change in absorption parametersdescribed above.

Optionally, the methods of the invention also comprise the step ofproviding to the patient in need thereof a therapeutically effectiveamount of tetrahydrobiopterin. The therapeutically effective amount willvary depending on the condition to be treated, and can be readilydetermined by the treating physician based on improvement in desiredclinical symptoms.

In one exemplary embodiment, such methods involve administering BH4 in adissolved form, wherein the formulation is dissolved in a liquidincluding but not limited to water, orange juice and apple juice. In oneexemplary embodiment, dissolved BH4 is administered to the patient in afasted condition, i.e., on an empty stomach. The invention furthercontemplates that the dissolved BH4, is administered at a specified timeincluding but not limited to morning, day, night, same time of the day,on an empty stomach, one or more times a day. In exemplary embodiments,the composition is administered to the patient when the stomach isempty, for example, at least 30 minutes, 45 minutes, or at least onehour before, and/or at least 90 minutes, or two hours, or 2.5 hours, orthree hours after a meal. Thus, BH4 may be ingested as a liquid productor pre-dissolved from a solid or semisolid dosage form prior toingestion. In a further embodiment, BH4 may also be dissolved in theoral cavity from a solid or semisolid dosage form prior to swallowingthe dissolved solution.

In another exemplary embodiment, such methods involve administering BH4in a solid dosage form including but not limited to tablets, capsules,candies, lozenges, powders, and granules, or semisolid form, includingbut not limited to oral sprinkle into jelly, that is swallowed withoutdissolving in a liquid including but not limited to water, orange juiceand apple juice, before swallowing. In one embodiment, swallowed BH4 isadministered to the patient in a fasted condition, i.e. on an emptystomach. The invention further contemplates that the BH4 swallowed as asolid or semisolid dosage form, is administered at a specified timeincluding but not limited to morning, day, night, same time of the day,on an empty stomach, one or more times a day. In exemplary embodiments,the composition is administered to the patient when the stomach isempty, for example, at least 30 minutes, 45 minutes, or at least onehour before, and/or at least 90 minutes, or two hours, or 2.5 hours, orthree hours after a meal.

In another embodiment, such methods involve administering BH4, whetherswallowed as a solid or semisolid dosage form, or dissolved in a liquid,with food, e.g. a high-fat food or a high-fat and/or high-calorie meal.The invention further contemplates that BH4, whether swallowed ordissolved, is administered at a specified time including but not limitedto morning, day, night, same time of the day, with food, e.g. a high-fatfood or a high-fat and/or high-calorie meal, one or more times a day. Inan exemplary embodiment, BH4 is ingested once daily as a solid dosageform just after meals. In a preferred embodiment the solid dosage formis a formulated tablet or capsule. In more exemplary embodiments, BH4 isingested within approximately 0 to 30 minutes, or 5 to 20 minutes, ofeating a meal. Regardless of whether it is ingested as a solid dosageform, liquid dosage form or as a dissolved solution, the in vivoexposure (or bioavailability) of BH4 is higher when ingested just aftermeals compared to fasting controls.

The BH4 and the food may be ingested at approximately the same time, orthe BH4 may be ingested before or after the food. The period of timebetween consuming the food and taking BH4, either swallowed ordissolved, may be at least 5 minutes. For example, BH4 may beadministered 60 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes,10 minutes, or 5 minutes before or after a meal.

In another embodiment, for some patients, e.g. adults, or some diseasestates, e.g. cardiovascular diseases or other diseases associated withNOS dysfunction, the methods of the invention involve administering anintact tablet rather than dissolving the tablet in a liquid, in order toimprove bioavailability.

In a second aspect, the invention contemplates a method of stabilizingBH4 in a patient's intestinal tract by decreasing intestinal pH, e.g.using proton exchange polymers. Corresponding products comprising BH4and acidifying excipients, such as proton exchange polymers, are alsocontemplated.

A third aspect of the invention contemplates a method of increasing gutresidence time for BH4, including but not limited to slowing of gutmotility using an agent which slows gut motility, such as a fatty acidand/or a glycerol fatty acid ester. Such hydrophobic agents can increasethe length of time that BH4 remains in the gut and can increase theamount of BH4 that gets absorbed. The length of time that BH4 remains inthe gut, when formulated with such agent(s), can be at least one and ahalf times, at least two times, at least three times, at least fourtimes, or at least five times longer than a BH4 formulation not havingsuch an agent. Suitable fatty acids include oleic acid, stearic acid,arachidic acid, palmitic acid, archidoic acid, linoleic acid, linolenicacid, erucidic acid, myristic acid, lauric acid, myristolic acid, andpalmitolic acid. Also contemplated to increase gut residence time forBH4 is inducement of gastric retention using alginic acid, andbioadhesion using polycarbophil. Corresponding products comprising BH4and agents that slow gut motility are contemplated.

A fourth aspect of the invention contemplates a method of modifying therelease of BH4 using a sustained release formulation such as HPMC,carbomer, etc. Corresponding products that are sustained releaseformulations are contemplated.

In a fifth aspect, the invention contemplates administering BH4 insterile liquid or sterile solid dosage form via routes other than oraladministration including but not limited to topical, intravenous,subcutaneous, intramuscular, intrathecal, ophthalmic, and inhalationalroutes of administration. Corresponding compositions and kits suitablefor such routes of administration, and methods of making the same, arecontemplated. For example, a transdermal or buccal patch for transdermalor buccal administration, respectively, comprising BH4 is contemplated.Sublingual tablets comprising BH4 are also contemplated. Suitable kitsare contemplated, including an inhaler device comprising BH4, or a kitcomprising BH4 and a dropper or sprayer.

One embodiment includes a liquid formulation of tetrahydrobiopterin(BH4) or a pharmaceutically acceptable salt thereof, including anaqueous solution of BH4 or pharmaceutically acceptable salt thereof, anantioxidant, and a pH buffer.

Another embodiment includes a method of making a liquid formulation oftetrahydrobiopterin (BH4) or a pharmaceutically acceptable salt thereof,including providing an aqueous solution containing BH4 orpharmaceutically acceptable salt thereof, adding an antioxidant and a pHbuffer to the solution containing BH4 or pharmaceutically acceptablesalt thereof, sparging the aqueous solution containing BH4 orpharmaceutically acceptable salt thereof, before or after addition ofantioxidant and pH buffer, with an inert gas or carbon dioxide, andsealing the sparged solution containing BH4 or pharmaceuticallyacceptable salt thereof, antioxidant, and pH buffer in a container.

In a sixth aspect, the invention contemplates an improved method ofmeasuring BH4 by utilizing tandem mass spectrometry and calculating theamount of reduced biopterin. Such methods can provide detection of BH4to a sensitivity for BH4 in the range of 5-1000 ng/mL, with an accuracyand precision as exemplified by a coefficient of variation (CV) % below15% (20% at the lower limit of quantitation, LLOQ). In an exemplaryembodiment, a method of measuring BH4 using HPLC(RP) coupled with tandemmass spectrometry (LC/MS/MS) comprises the steps of: (1) subjectingsamples of blood, plasma, tissue homogenates, or urine to oxidation; (2)subjecting the oxidized samples to iodometry; (3) passing said oxidizedsamples through an ion exchange column; (4) measuring total and oxizedbiopterin in said samples using HPLC and tandem mass spectrometry; andcalculating the amount of reduced biopterin as the difference betweensaid total biopterins less said oxidized form. In one embodiment,samples are treated with acidic oxidation, wherein the method comprisesthe steps of (1) treating said samples with KCl, HCl or TCA; (2)subjecting said acid-oxidized samples to iodometry; (3) running saidoxidized samples through an ion exchange column; (4) measuring totalbiopterin comprising 6R-BH4, R-q-DHBP (which is immediately reduced invivo to 6R-BH4 such that the measured reduced biopterin is based mainlyupon 6R-BH4), DHBP, and BP in said samples using HPLC and tandem massspectrometry. In another embodiment, samples are treated by alkalineoxidation, wherein the method comprises: (1) treating said samples withKI, I or NaOH; (2) subjecting said alkaline oxidized samples toacidification with HCl or TCA; (3) subjecting said oxidized samplesiodometry; (4) running said samples through an ion exchange column; (5)measuring oxidized biopterin comprising DHBP and BP using HPLC andtandem mass spectrometry; and (6) calculating the amount of reducedbiopterin (6R-BH4+R-q-DHBP) as the difference between total biopterinsless the oxidized form.

Another aspect of the invention is a mobile phase solution forreverse-phase HPLC separation of dihydrobiopterin, biopterin, andanalogs thereof, including an aqueous solution including methanol,sodium acetate, citric acid, EDTA, and 1,4-dithioerythritol. Similarlycontemplated is a method of separating dihydrobiopterin and biopterin,or analogs thereof, from a mixture containing both base and dihydroforms, including performing reverse phase HPLC using a mobile phasecomprising an aqueous solution including methanol, sodium acetate,citric acid, EDTA, and 1,4-dithioerythritol, on a mixture containingdihydrobiopterin and biopterin, or an analog of dihydrobiopterin and ananalog of biopterin.

Another aspect of the invention is a method of quantitating biopterinsin a mixture of biopterin species, including providing a mixturecomprising biopterin and at least one of dihydrobiopterin andtetrahydrobiopterin, or analogs of biopterin and at least one ofdihydrobiopterin and tetrahydrobiopterin, separating the biopterinspecies in the mixture by reverse phase HPLC, and in the case oftetrahydrobiopterin and analogs thereof, performing electrochemicaldetection by oxidizing the tetrahydrobiopterin and analogs thereofpresent by a first electrode to quinonoid dihydrobiopterin forms,followed by reducing the quinonoid forms back to tetrahydrobiopterin andanalogs thereof present at a second electrode, and measuring currentgenerated by the reduction reaction to determine the concentration ofspecies, and/or in the case of dihydrobiopterin, analogs thereof,biopterin, or analogs thereof, measuring such species by fluorescencedetection following post-column oxidation of dihydrobiopterin species tobiopterin.

For the compositions and methods described herein, preferred components,and compositional ranges thereof, can be selected from the variousexamples provided herein.

Other features and advantages of the invention will become apparent fromthe following detailed description. It should be understood, however,that the detailed description and the specific examples, whileindicating preferred embodiments of the invention, are given by way ofillustration only, because various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a powder X-ray diffraction pattern characteristic ofcrystal polymorph form B of 6R-(L-erythro)-5,6,7,8-tetrahydrobiopterin.

FIG. 2 is a graph of the characteristic X-ray diffraction patternexhibited by form A of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 3 is a graph of the characteristic X-ray diffraction patternexhibited by form F of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 4 is a graph of the characteristic X-ray diffraction patternexhibited by form J of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 5 is a graph of the characteristic X-ray diffraction patternexhibited by form K of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 6 is a graph of the characteristic X-ray diffraction patternexhibited by hydrate form C of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 7 is a graph of the characteristic X-ray diffraction patternexhibited by hydrate form D of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 8 is a graph of the characteristic X-ray diffraction patternexhibited by hydrate form E of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 9 is a graph of the characteristic X-ray diffraction patternexhibited by hydrate form H of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 10 is a graph of the characteristic X-ray diffraction patternexhibited by hydrate form O of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 11 is a graph of the characteristic X-ray diffraction patternexhibited by solvate form G of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 12 is a graph of the characteristic X-ray diffraction patternexhibited by solvate form I of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 13 is a graph of the characteristic X-ray diffraction patternexhibited by solvate form L of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 14 is a graph of the characteristic X-ray diffraction patternexhibited by solvate form M of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 15 is a graph of the characteristic X-ray diffraction patternexhibited by solvate form N of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

FIG. 16 is a flow chart for the measurement of biopterin.

FIG. 17 is a summary of the validation of the biopterin assay.

FIG. 18 is a table showing pharmacokinetic parameters of totalbiopterins in plasma after a single oral administration of sapropterin(BH4) to rats.

FIG. 19 shows plasma biopterin concentration and reduced-form ratioafter single-dose administration of sapropterin (BH4) to rats.

FIG. 20 shows plasma biopterin concentration and reduced-form ratioafter a single-dose administration of sapropterin (BH4) in monkeys.

FIG. 21 is a table showing pharmacodynamic parameters of totalbiopterins in plasma after single-dose administration of sapropterin(BH4) to monkeys.

FIG. 22 shows the schedule of events for the evaluation of safety.

FIG. 23 shows the mean plasma concentrations of BH₄ after oraladministration of 10 mg/kg of BH4 as dissolved and intact tablets underfasted conditions and intact tablets under fed conditions to healthyvolunteers—linear axes.

FIG. 24 shows the mean plasma concentrations of BH₄ after oraladministration of 10 mg/kg of BH4 as dissolved and intact tablets underfasted conditions and intact tablets under fed conditions to healthyvolunteers—semi-logarithmic axes.

FIG. 25 shows a table summarizing the pharmacokinetic parameters for BH₄after oral administration of 10 mg/kg of BH4 as dissolved and intacttablets under fasted conditions and intact tablets under fed conditionsto healthy volunteers.

FIG. 26 shows a statistical comparison of pharmacokinetic parameters forBH₄ after oral administration of 10 mg/kg of BH4 as dissolved and intacttablets under fasted conditions and intact tablets under fed conditionsto healthy volunteers.

FIG. 27 shows a stability study of BH4 formulated with 5% mannitol in anaqueous solution both before and after two weeks stored at −20° C.

FIG. 28 shows a dissolution profile of a BH4 capsule formulation bothbefore and after storage for 54 days at 40° C.

FIG. 29 shows a dissolution profile of two BH4 formulations—a BH4bioadhesive tablet and BH4 bioadhesive granules.

FIG. 30 shows a dissolution profile of various sustained releaseformulations of BH4.

FIG. 31 shows a dissolution profile of various sustained releaseformulations of BH4.

FIG. 32 shows a schematic diagram of a floating dosage formulations ofBH4.

FIG. 33 shows a dissolution profile of various floating dosageformulations.

FIG. 34 shows a schematic diagram of gas generating dosage forms of BH4.

FIG. 35 shows a pharmacokinetic profile of various BH4 formulations.

FIG. 36 shows a stability study of intravenous BH4 formulations at pH 4over 35 days.

FIG. 37 shows a stability study of various intravenous BH4 formulationsover 350 hours.

FIG. 38 shows a stability study of intravenous BH4 formulations atvarious BH4 concentrations.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides improved methods of orally administering apurified preparation of 6R-(L-erythro)-5,6,7,8-tetrahydrobiopterin,including a pharmaceutically acceptable salt thereof. The invention isbased on the finding that orally administered tetrahydrobiopterin (BH4)has low gastrointestinal absorption, which is a major contributingfactor to the low bioavailability of BH4.

The chemical structure of 6R-(L-erythro)-5,6,7,8-tetrahydrobiopterin(BH4) is shown below:

Tetrahydrobiopterin is a water soluble organic compound with low lipidsolubility. Based on an in silico experimental analysis using BioLoomsoftware (version 1.5 from Biobyte Corp in Claremont Calif.), theoctanol-water partition coefficient of BH4 was determined to be −1.17.Optimal penetration of biological membranes as approximated by theoctanol/water partition coefficient occurs at around a log P of 2 or100-× fold higher lipid solubility. Although a low ClogP allows thissubstrate to solubilize readily under physiological conditions, theability of the substrate to penetrate bilipid layers within biologicalmembranes is restricted, which may limit oral availability.

In vivo studies in rats and monkeys described herein showed that only8-11% of BH4 is absorbed in the gut with the majority being excreted inthe feces when compared to intravenous administration of BH4 at similardoses. Such variability in absorption of BH4 was also shown in a studydescribed herein on the effect of food on the bioavailability of BH4 inhealthy humans. Although the administration of BH4 in water and orangejuice under fasted conditions resulted in comparable mean plasmaconcentrations and mean values for Cmax and AUC(0-t), the administrationof BH4 concurrent with a high fat, high caloric meal resulted in asignificant increase in the mean plasma concentrations and mean valuesfor Cmax and AUC(0-t) when BH4 was administered in water.

Although there is ample literature describing increased bioavailabilityin fed conditions, this food effect is typically seen with lipophilic(i.e., lipid soluble) water-insoluble drugs and not usually with highwater soluble active substance such as BH4. The usual explanation forincreases in bioavailability under fed conditions for lipophiliccompounds is that high fat meals help solubilize the drug since “likedissolves like” and this makes it available for absorption. Anotherpossible explanation is that high fat meals stimulate the secretion ofbile acids which are natural bio-surfactants that help solubilize andemulsify the fats we eat to aid their digestion. These bile acids arealso thought to solubilize water-insoluble compounds thereby making themavailable for absorption. However, BH4 does not need solubilization tobe absorbed since its solubility is greater than 1000 mg/mL and thecompound is one of the most soluble drugs known. Therefore theenhancement of its bioavailability by high fat, high-energy meals is notconsistent with such known mechanism.

However, administration as a solid or semi-solid dosage form and/or witha high-fat meal may maximize bioavailability by increasing the residencetime of BH4 in the acidic milieu of the stomach and uppergastrointestinal tract (GIT) where BH4 is chemically stable. Thestability of BH4 decreases with increasing pH and its half-life in pH6.8 buffer solution, which is roughly the pH of the small intestine, isabout 15 minutes. At pH 3.1, which is within realm of the typical pH ofthe stomach in normal volunteers, the stability of BH4 at aconcentration of 1 mg/mL is over 3 hours. The chemical stability of BH4may further increase when the pH of the stomach drops below pH 3.1.Therefore prolonged stomach residence time provides intact drug to thestomach wall for absorption, whereas rapid emptying into the intestinedegrades BH4 and is thus not available to be absorbed.

Thus, to maximize oral bioavailability of BH4 at each administration,BH4 should be taken with food, e.g., a high fat food or a high fatand/or high calorie meal. Alternatively, to maximize consistency of oralbioavailability between administrations, BH4 should be taken on an emptystomach (e.g., 1 hour before or 2 hours after a meal).

As used herein, the term “bioavailability” refers to the fraction of anadministered dose of a drug entering systemic circulation. If the drugwere administered intravenously, then its bioavailability theoreticallywould be 100%. However, if the drug were administered via other routes(such as orally), then its bioavailability would be less than 100% as aresult of, for example, incomplete absorption in the GI tract,degradation or metabolism prior to absorption, and/or hepatic first passeffect.

The term “high fat meal” refers generally to a meal of at least about700 kcal and at least about 45% fat (relative percentage of kcal whichare fat), or alternatively at least about 900 kcal and at least about50% fat. The term “high fat food” refers generally to a food comprisingat least 20 g of fat, or at least 25, 30, 35, 40, 45, or 50 g of fat,and/or at least about 45% or 50% fat. One FDA Guidance defines a“high-fat meal” as approximately 50% of total caloric content of themeal, whereas a “high-calorie meal” is approximately 800 to 1000calories. The FDA recommends a high-fat and high-calorie meal as a testmeal for food-effect bioavailability and fed bioequivalence studies.This test meal should derive approximately 150, 250, and 500-600calories from protein, carbohydrate and fat, respectively. An exampletest meal consists of two eggs fried in butter, two strips of bacon,four ounces of hash brown potatoes and eight ounces of whole milk.Substitution is possible if a similar amount of calories from protein,carbohydrate, and fat has comparable meal volume and viscosity (Guidancefor Industry, Food-Effect Bioavailability and Fed BioequivalenceStudies, U.S. Department of Health and Human Services, Food and DrugAdministration, Center for Drug Evaluation and Research (CDER), December2002).

In a first aspect, the invention provides methods of orallyadministering a purified preparation of6R-(L-erythro)-5,6,7,8-tetrahydrobiopterin (BH4), including apharmaceutically acceptable salt thereof.

In some embodiments, the methods involve informing the patient thatadministration of tetrahydrobiopterin with food has an effect onpharmacokinetics. In an exemplary embodiment, the methods comprise thestep of informing the patient that absorption of tetrahydrobiopterin isincreased when it is ingested with food compared to when ingestedwithout food. In some embodiments, the patient is informed thatingestion shortly following a meal, for example, a high-fat,high-calorie meal, results in an increase in any one, two, three or allof the following parameters: mean plasma concentration, Cmax, AUC,AUC(0-t) and/or AUC(inf). In exemplary embodiments, the patient isinformed that administration of BH4 with a high-fat meal increases Cmaxand AUC compared to administration of BH4 without food (in a fastingcondition). In some embodiments, the relative increase can be at least20% or 30% or more.

In alternative embodiments or in addition to the preceding embodiments,the method of administering tetrahydrobiopterin comprises informing thepatient that absorption of tetrahydrobiopterin is increased wheningested as an intact tablet compared to when ingested after beingdissolved in liquid. In some embodiments, the patient is informed thatingestion of intact tablets results in an increase in any of thefollowing parameters: mean plasma concentration, Cmax, AUC, AUC(0-t) orAUC(inf). In exemplary embodiments, the patient is informed thatadministration of BH4 as an intact tablet increases Cmax and AUCcompared to administration of BH4 after being dissolved in a liquid. Insome embodiments, the relative increase can be at least 20% or more.

Any of the preceding methods may be carried out by providing oradministering tetrahydrobiopterin in a container containing printedlabeling informing the patient of the change in absorption parametersdescribed above.

Optionally, the methods of the invention also comprise the step ofproviding to the patient in need thereof a therapeutically effectiveamount of tetrahydrobiopterin. The therapeutically effective amount willvary depending on the condition to be treated, and can be readilydetermined by the treating physician based on improvement in desiredclinical symptoms.

In one exemplary embodiment, such methods involve administering BH4 in adissolved form, wherein the formulation is dissolved in a liquidincluding but not limited to water, orange juice and apple juice. In oneembodiment, dissolved BH4 is administered to the patient in a fastingcondition, i.e., on an empty stomach. The invention further contemplatesthat the dissolved BH4, is administered at a specified time includingbut not limited to morning, day, night, same time of the day, on anempty stomach, one or more times a day. In exemplary embodiments, thecomposition is administered to the patient when the stomach is empty,for example, at least 30 minutes, 45 minutes, or at least one hourbefore, and/or at least 90 minutes, or two hours, or 2.5 hours, or threehours after a meal. Thus, BH4 may be ingested as a liquid product orpre-dissolved from a solid or semisolid dosage form prior to ingestion.In a further embodiment, BH4 may also be dissolved in the oral cavityfrom a solid or semisolid dosage form prior to swallowing the dissolvedsolution.

These approaches maximize absorption rate and bioavailability byensuring that BH4 is fully dissolved in solution or biologic fluidsbefore it is delivered to its absorption sites, which are primarily thestomach and the intestine. Dissolution of active pharmaceuticalingredients or drug in solution is a prerequisite to absorption into thesystemic (blood and lymphatic) circulation. When solid dosage forms suchas tablets and capsules are administered orally, they go through asequential series of steps such as disintegration into granules,de-aggregation into powders and dissolution prior to absorption into thesystemic circulation. These series of steps are bypassed byadministering liquid, semisolid and fast dissolving solid dosage forms.Thus the active substance is available earlier for absorption, andbecause there is no guarantee that a solid dosage form will release allthe active substance contained within it before it transits through theabsorptive sites, the formulations in which the active substance ispresent in dissolved form before it reaches the absorptive sites usuallyexhibits the greater bioavailability.

These dosage forms reduce variability in blood levels because thevariability is dosage form disintegration and dissolution in vivo in thehuman is obviated. The rate of in vivo disintegration and dissolution ofa sold dosage form of BH4 targeted for immediate-release in the stomachdepends on the human-to-human variability in the pH of the gastricfluid—fed and unfed (fasting)—and the strength of the agitationintensity of the stomach as determined by the strength of gastricmotility and gastric emptying rates into the small intestine. Sinceliquid, semisolid, lozenge/candy and fast dissolving solid dosage formsdo not have to be subjected to disintegration and dissolution, theirblood levels are less variable than when BH4 is given as immediaterelease solid dosage forms (tablets and capsules).

In another exemplary embodiment, such methods involve administering BH4in a solid dosage form including but not limited to tablets, capsules,candies, lozenges, powders, and granules, or semisolid form, includingbut not limited to oral sprinkle into jelly, that is chewed or swallowedwithout dissolving in a liquid including but not limited to water,orange juice and apple juice, before swallowing. In one embodiment,swallowed BH4 is administered to the patient in a fasting condition,i.e., on an empty stomach. The invention further contemplates that theBH4 swallowed as a solid or semisolid dosage form, is administered at aspecified time including but not limited to morning, day, night, sametime of the day, on an empty stomach, one or more times a day. Inexemplary embodiments, the composition is administered to the patientwhen the stomach is empty, for example, at least 30 minutes, 45 minutes,or at least one hour before, and/or at least 90 minutes, or two hours,2.5 hours, or three hours after a meal.

In another embodiment, such methods involve administering BH4, whetherswallowed as a solid or semisolid dosage form, or dissolved in a liquid,with food, e.g. a high-fat food or a high-fat and/or high-calorie meal.The invention further contemplates that BH4, whether swallowed ordissolved, is administered at a specified time including but not limitedto morning, day, night, same time of the day, with food, e.g. a high-fatfood or a high-fat and/or high-calorie meal, one or more times a day. Inan exemplary embodiment, BH4 is ingested once daily as a solid dosageform just after meals. In a preferred embodiment the solid dosage formis a formulated tablet or capsule. In more exemplary embodiments, BH4 isingested within approximately 0 to 60 minutes, approximately 0 to 30, or5 to 20 minutes of eating a meal. Regardless of whether it is ingestedas a solid dosage form, liquid dosage form or as a dissolved solution,the in vivo exposure (or bioavailability) of BH4 is higher when ingestedjust after meals compared to fasting controls.

The BH4 and the food may be ingested at approximately the same time, orthe BH4 may be ingested before or after the food. The period of timebetween consuming food, e.g., a high-fat food or a high-fat and/orhigh-calorie meal and taking BH4 either swallowed or dissolved may be atleast 5 minutes. BH4 may be administered 60 minutes, 30 minutes, 25minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes afteringestion of a meal.

In another embodiment, for some patients, e.g. adults, or some diseasestates, e.g. cardiovascular diseases or other diseases associated withNOS dysfunction, the methods of the invention involve administering anintact tablet rather than dissolving the tablet in a liquid, in order toimprove bioavailability.

Administration of BH4 according to the methods of the invention resultsin mean plasma concentrations and/or rate of gastrointestinal absorptionand/or mean values for Cmax and/or AUC(0-t) and/or AUC (inf) thatexceeds the values when BH4 is administered under fasted conditions.

Administration of an intact tablet under fasted conditions resulted inan average 20% increase in Cmax and AUC relative to dissolved tablets.Administration of a dissolved tablet in either water or orange juice oran intact tablet after a high fat/high calorie meal resulted inincreases in Cmax and AUC that ranged from approximately 30% (intacttablet) to 80% (water). Administration of BH4 as an intact tabletfollowing a high fat and high calorie meal resulted in an approximate30% increase in the extent of absorption compared to administrationwithout food. Administration of BH4 as an intact tablet resulted in anapproximate 20% increase in the extent of absorption compared toadministration of dissolved tablets.

“Mean plasma concentration” means the average of readings ofconcentration in a series of plasma samples.

“Cmax” means the maximum observed plasma concentration.

“AUC” means the area under the plasma concentration-time curve.

“AUC_(0-t)” means the area under the plasma concentration-time curvefrom time 0 to the time of the last measurable concentration.

“AUC_((inf))” means the calculated area under the plasmaconcentration-time curve from time 0 to infinity.

The “rate of gastrointestinal absorption” of BH4 is estimated from thearea under the plasma total biopterin concentration increase (ΔCp)-timecurve (ΔAUC) after the administration of BH4 using the followingformula:

Absorption rate (%)=(ΔAUC after p.o. dose/ΔAUC after i.v. dose)×(i.v.dose/p.o. dose×100)

Preferably at least 99.5% pure 6R-BH4 is used. Any salt, including thedihydrochloride salt, and any crystalline form of BH4 may be utilizedaccording to the methods and compositions of the invention. A variety ofsalts and crystalline forms are described in U.S. Patent Publication No.2006/0040946, incorporated herein by reference in its entirety, and/orthe stable solid formulation described in Int'l Publication No. WO06/55511, also incorporated herein by reference in its entirety. Thevarious crystalline forms may conveniently be formed into a tablet,powder or other solid for oral administration.

In a second aspect, the invention contemplates a method of stabilizingBH4 by decreasing intestinal pH using proton exchange polymers. BH4 isadministered orally daily as a solid or liquid dosage form comprisinginactive ingredients that enhance the stability of BH4 beyond thestomach by lowering the pH of the intestine and thus preserving BH4 frombeing oxidized rapidly. Since BH4 is more stable in acidic media than inbasic media, acidifying excipients/inactive ingredients are included insolid dosage (tablets, capsules, etc) formulations of BH4 to lower thepH of the intestinal fluids and thereby enhance the chemical stability.The larger area or window of the gastrointestinal tract (GIT) availablefor absorption optimizes the consistency of absorption by expanding thecurrent limited window of absorption believed to be limited to thestomach and the duodenum to the intestine. Such dosage forms include butare not limited to effervescent tablets, powders and granules (to beresuspended in liquid before administration) and acidifier materials.Unlike small molecule acids, bulky polymeric acids remain in the GITlonger and are not absorbed by the GIT, but donate their protons to theGIT fluids to lower the environmental pH. Examples ofexcipients/inactive ingredients that comprise the formulation arecarboxylic acid small molecules such as maleic, fumaric and citric acidsor inorganic small molecules such phosphoric acid, acetic acid and theirsalt forms. Other examples are pharmaceutically acceptable acids such aspolymeric carboxylic acid classes including polymethacrylic acids,carbomers, polycarbophil, Eudragits, acid forms of crosscarmelose andstarch glycolic acid, etc. The formulations also contain additionalexcipients to enhance stability such as anti-oxidants (e.g., thiols suchcysteine, N-acetyl cysteine, etc; ascorbic acid; methionine; etc.) andother excipients known in the trade to enable manufacturability andenhance the quality and performance attributes of the formulation.

A third aspect of the invention contemplates a method of increasing gutresidence time for BH4, including but not limited to slowing of gutmotility using an agent which is capable of slowing gut motility of BH4,such as a fatty acid and/or a glycerol fatty acid ester. Fatty acids caninclude oleic acid, stearic acid, arachidic acid, palmitic acid,archidoic acid, linoleic acid, linolenic acid, erucidic acid, myristicacid, lauric acid, myristolic acid, and palmitolic acid. Alsocontemplated for increasing gut residence time of BH4 are the inducementof gastric retention using alginic acid and bioadhesion usingpolycarbophil. In one embodiment, dosage forms of BH4 are administeredas oral buoyant formulations that float and release BH4 in a definedfashion in the gastric fluid and are retained longer in the stomachbecause they are more resistant to gastric emptying from the stomachthan formulations that are non-buoyant or dissolve rapidly in thestomach. This design approach is based on gastro-retention of the dosageform via the use of a gas-generating excipient within the dosage form,low-density excipients that render the dosage form buoyant in GIT fluidsor a combination of a gas and low-density materials in a dosage form toenable the floating of the dosage form in the fluid contents of the GIT.Prolonged retention and release of the dosage form in the stomach milieuwherein BH4 is more stable in its acidic fluids will enhance bothresidence time of the dosage form in the stomach and the stability ofBH4 and thus make BH4 available for a longer period absorption in thestomach and duodenum than standard tablet and capsule dosage forms.Formulations of BH4 will comprise of one or more antioxidants,excipients known in the field to enable manufacturing anddisintegration/dissolution of the solid dosage form and additionalexcipients that generate a gas or mixture of gases (e.g., carbondioxide) upon contact of the formulation with aqueous media and or GITfluids. Water-soluble antioxidants are preferred, for example, ascorbicacid, methionine, and thiols (cysteine, N-acetyl cysteine andglutathione) or anti-oxidants that are converted to a solubleantioxidant in the GIT, e.g., ascorbyl palmitate which is converted toascorbic acid in the GIT. Excipients added to the formulation includecarbonates and bicarbonates that react directly with BH4 to form carbondioxide and small and polymeric acids described previously to react withthe carbonates and bicarbonates to produce additional carbon dioxide asneeded.

In another embodiment, dosage forms of BH4 are administered that adherefor a prolonged time to the mucous surfaces of the GIT (i.e.,bioadhesive formulation), preferably in, but by no means limited to thestomach where due to the acidity of gastric fluids, BH4 is more stablethan in the intestine. BH4 is released in a controlled manner from thebioadhesive dosage form. The solid dosage form is designed to containBH4, one or more antioxidants, excipients known in the field to enablethe manufacturing of quality dosage forms and control thedisintegration/dissolution of the dosage form and a bioadhesive additivesuch as polycarbophil in its free acid form or as a salt form. Otherpolymeric acids such as polymethacrylic acids, carbomers and cellulosederivatives, e.g., HPMC, HPC, etc. may be combined with or substitutedfor polycarbophil. The antioxidants are preferably soluble, for example,ascorbic acid, methionine, cysteine, N-acetyl cysteine and glutathioneor can be converted to a soluble antioxidant such as ascorbic acid inthe GI T, e.g., ascorbyl palmitate. In one embodiment, the components ofthe formulation are blended together and manufactured as a solid dosageform, e.g., tablets or capsules. The solid dosage form may be entericcoated to deliver BH4 past the stomach into the intestine or not entericcoated designed to release BH4 in the stomach. In another embodiment,the components of the solid dosage form may be subdivided into differentportions and the various portions are blended separately before they areprocessed to form multilayered dosage forms. The multilayered dosageform may contain the bioadhesive and a few excipients in the outermostlayer of a tablet, wrapped around other layers that contain BH4 (i.e.,active region inside a bioadhesive envelope) or as a wrap-aroundcylindrical plug filled into a capsule wherein one or more other layersare assembled beneath or within the bioadhesive envelope. Alternatively,the bioadhesive and other layers in the tablet or capsule plugs may belayered in a parallel bi- or multilayer configuration. These designsallow the bioadhesive to interact with the GI membrane or GI membranemucus to anchor the dosage form to the membrane slowing down its transitthrough the GI tract and thus increasing residence time. Such dosageforms may also be enteric coated. Yet another embodiment of the methodused with BH4 is to employ polymeric inactive ingredients (excipients)with functional groups that bind to GIT mucus to delay the transit ofthe dosage form through the GIT. Dosage forms of BH4 are formulated withthiolated polymer excipients (polymer-SH) such aspolycarbophil-cysteine, polypolymethacrylic acid-cysteine, carboxymethylcellulose-cysteine, chitosan derivatives-cysteine, etc. These thiolatedpolymers confer both bioadhesive and anti-oxidant properties on BH4considerably enhancing absorption. Other excipients included in theseformulations are antioxidants and performance and manufacture-aidingexcipients.

In yet another embodiment, oral dosage forms containing inactiveexcipients or active ingredients are used to slow gastric motility.Slowing down the transit of BH4 dosage form through the GIT tract willincrease the residence time of the molecule and thus enable a largerfraction of the administered dose to be absorbed. Generally regarded assafe (GRAS) excipients employed in oral formulations to delay gastricemptying and/or delay intestinal motility preferably comprise dietaryfats such as fatty acids, glycerides of fatty acids, and derivatives offatty acids and glycerides such as Cremophor™ (polyoxyl castor oilderivatives), etc. Active excipients include agents that slow gutmotility such as general or selective (M₃) antimuscarinic oranticholinergic agents.

A fourth aspect of the invention contemplates a method of modifying therelease of BH4 using a sustained release formulation such as HPMC,carbomer, etc. This concept comprises delivering BH4 dosage forms to theGI tract by modifying or altering the release of BH4 fromimmediate-release to slow, prolonged, controlled and or timed release.Slow, prolonged and controlled release is achieved using excipientsknown in the art and BH4 is protected within the delivery system fromchemical degradation by the presence of stability enhancers such asanti-oxidants. Such methods can maximize bioavailability since BH4 isstabilized within the formulation and in the environment surrounding theformulation to enable the active molecule to absorbed intact into thesystemic circulation as the formulation transits the entire length ofthe GIT. This approach provides a larger window of the GIT forabsorption and does so by preventing the degradation of BH4 in thehigher pH milieu so that BH4 is available to be absorbed. Antioxidantswill be included in the formulation to protect the drug from degradingin intestinal fluids due to near neutral pH of the intestinal fluids.Slow, prolonged and controlled delivery will also deliver BH4 to lowoxygen tension regions of the GIT. Timed release is achieved usingexcipients known in the art such as pH sensitive polymers that dissolveonly when the pH reaches a value wherein the polymer is soluble.

In another embodiment, the invention contemplates enteric coating of theBH4 dosage form to ascertain whether including acidic excipients in aformulation of BH4 does indeed increase absorption of BH4 by loweringthe pH of the intestine and thus stabilizing BH4 in the intestine to beavailable for absorption. Thus, enteric coating will be used to keep theexcipients and drug together at the site where the excipient is expectedto protect BH4. If the BH4 dosage form were allowed to disintegrate inthe stomach, the acidic excipients may not empty together into thestomach and may not provide protection.

Enteric coating protects compounds susceptible to acid-catalyzeddegradation in the stomach from getting degraded by the acid in thestomach. Enteric coating materials prevent the tablet or capsule fromreleasing the active compound in the stomach because the enteric coatingmaterials are insoluble in acid. Once the enteric-coated dosage formreaches the intestine where the pH value varies from pH 5-8, thematerials become soluble and release the active substance in theintestine. In contrast, sustained release formulations are designed torelease medicaments over as long a length/area of the GIT as possible.Coating a sustained release formulation to release just past the stomachmay be necessary only if the medicaments contained in it areacid-labile.

In a fifth aspect, the invention contemplates administering BH4 insterile liquid or sterile solid dosage form via routes other than oraladministration including but not limited to topical, intravenous,subcutaneous, intramuscular, intrathecal, ophthalmic, and inhalationalroutes of administration. BH4 is formulated as a sterile liquid or soliddosage form at the appropriate concentration desired.

The advantages of a sterile liquid dosage form of BH4 for intravenousadministration may include: (1) more predictable kinetics, with thepotential for higher serum levels; (2) no requirement of a functionalgastrointestinal tract; (3) no requirement for patient participation;and (4) absence of a noncompliance concern. Intravenous formulations ofBH4 may be particularly beneficial in managing conditions requiringexpedited delivery of fluids and medications throughout the body or tobody compartments normally difficult to access via oral or other formsof administration, including but not limited to rabies, meningitis,organ transplantation/preservation, sub-arachnoidal hemorrhages, braintrauma, stroke, coronary artery bypass surgery, cerebrovascularvasospasm, blood transfusion/preservation, pulmonary hypertension,sickle cell disease, pre-eclampsia, and post-chemotherapy vasculardisease.

BH4 is highly susceptible to oxidation in aqueous solution and inphysiologic aqueous pH solutions (Davis, et al., Eur. J. Biochem. 173,345-351 (1988); Kirsch, et al., J. Biol. Chem. 278, 24481-24490 (2003)).Most determinations of BH4 stability have been carried out in neutral tomildly alkaline pH 7.4 solutions to mimic the likely stability behaviorof BH4 under physiologic plasma pH condition. Although European PatentApplication No. 1 757 293 A discloses liquid or syrup formulations, suchformulations consist of solid state powder blends or granulations thatrequire reconstitution with water prior to oral ingestion. The presentaspect of invention contemplates liquid formulations not limited topowders or granulations for constitution. The invention alsocontemplates compounded liquid formulations able to remain stable atambient temperature for a sufficient period of time to allow processingin sterile product fill/finish facilities to be filled into ampoules,bottles or vials as a liquid product or filled into vials to befreeze-dried into lyophilized products.

The liquid and lyophilized formulations for reconstitution can also bedelivered via the nasal, ophthalmic and ear canal for therapeuticeffects. The formulation of a lyophilized product requires priordissolution of BH4 in a liquid, preferably aqueous, and the processingof the liquid product in a sterile facility (i.e. compounding, sterilefiltration and filling of the sterile-filtered liquid into vials priorto the loading of the filled vials into a lyophilizer forlyophilization). Maintaining the stability of solubilized BH4 duringsterile processing and preventing its degradation are key prerequisitesto manufacturing lyophilized product that satisfies impuritiesspecification for the fill-finished product. Therefore the compositionof the lyophilized product contains appropriate stabilizers thatminimize or obviate BH4 degradation during the fill finish process. Theformulations described herein would stabilize BH4 solutions duringsterile fill/finish manufacturing, a process that takes a minimum of sixhours, and also provide commercially stable product.

The formulations include BH4, preferably in concentration in a range of0.1 mg/mL to 10 mg/mL. Due to the high solubility of BH4, formulationswith concentrations up to about 100 mg/mL, for example, can also beprepared. The general relative compositional makeup and methodsdescribed herein are applicable for making highly concentratedsolutions.

Liquid formulations of BH4 preferably are formulated in pH 1 to 8 buffersolutions, preferably in pH 2 to 7 buffer solutions. The pH bufferschosen are buffer compounds capable of providing substantial bufferingcapacity at a particular pH desired, as judged by how close the bufferionization constant or constants are to the desired pH of the liquidformulation. Thus any buffer compounds may be employed as long as one ormore of the compound's ionization constants are close to the desired pHof the formulation. Examples of buffers that may be employed in the pH1-8 range comprise various acids/bases and their respective conjugateacids/bases or salt forms, including but not limited to: hydrochloricacid (pH 1-2), maleic acid (pH 1-3), phosphoric acid (pH 1-3), citricacid (pH 3-6), acetic acid (pH 4.7±1.0), sodium phosphate dibasic (pH6-8), tromethamine (TRIS, pH 8.3±1.0), and the like.

Intravenous Formulations

Intravenous formulations are stabilized using an anti-oxidant or acombination of 2 or more antioxidants. Combinations of anti-oxidants canbe synergistic in obviating instability of the formulation. Spargingwith inert gases and or carbon dioxide to remove dissolved oxygen fromsolution is optional, but is preferred when low concentrations ofantioxidants are used, and further preferably when both lowconcentrations of BH4 and antioxidants are used. Stabilization of BH4 inaqueous solution is influenced by the interactions of the concentrationof BH4 with the antioxidant and pH. Thus, for example, highconcentrations of BH4 require less antioxidant concentrations than lowconcentrations of BH4. Furthermore, BH4 is more stable at low pH than athigh pH. Therefore desired high pH formulations preferably have higherantioxidant concentrations, more preferably a combination of 2, 3, ormore antioxidants, and still further preferably sparging withnon-oxidizing gas (e.g., inert gas or carbon dioxide) followed byhermetically or near-hermetically sealing the primary container in anatmosphere of a non-oxidizing gas (e.g., inert gas or carbon dioxide) tofurther enhance the stability of the drug product.

Example ranges for BH4 liquid formulations are given in Tables 1 and 2.Formulated or compounded solutions are optionally sparged with an inertgas (e.g., argon or nitrogen) or carbon dioxide in the compounding tankand primary containers preferably are sealed in a blanket of inert gasor carbon dioxide to remove oxygen from the container headspace. Theformulation can be scaled up to any volume by multiplying the componentamounts by an appropriate scale up factor.

TABLE 1 General examples of composition ranges in a low pH (e.g., pH4.0) formulation Amount % Weight/ Components (mg) Volume Function BH40.10-100   0.01-10.00 Active substance L-Cysteine 0.00-50.00  0.00-5.00 Antioxidant Ascorbic Acid 0.00-500.00 0.00-50.00 Antioxidant SodiumMetabisulfite 0.00-300.00 0.00-30.00 Antioxidant Citric Acid 0.26-19.87 0.03-1.99  Buffering agent Sodium Citrate, Dihydrate 2.57-192.750.26-19.27 Buffering agent Water for Injection qs 1.00 mL 1.00 mLDiluent

TABLE 2 General examples of composition ranges of a neutral pH (e.g., pH7.0) formulation Amount % Weight/ Components (mg) Volume Function BH40.10-100   0.01-10.00 Active substance L-Cysteine 0.00-50.00  0.00-5.00 Antioxidant Ascorbic Acid 0.00-500.00 0.00-50.00 Antioxidant SodiumMetabisulfite 0.00-300.00 0.00-30.00 Antioxidant Sodium Monobasic0.50-11.02  0.05-1.02  Buffering agent Phosphate, Monohydrate SodiumDibasic Phosphate 0.44-17.80  0.04-1.78  Buffering agent Water forInjection qs 1.00 mL 1.00 mL Diluent

The antioxidants employed for liquid formulations preferably areselected from one or more of thiol-based (e.g., L-cysteine), ascorbicacid and sulfite-based (e.g. sodium metabisulfite) compounds. Solutionspreferably are sparged with inert gases or carbon dioxide to expeloxygen from the BH4 solutions and then hermetically sealed in ampoulesor hermetically capped vials and bottles using metallic beveragebeer-type caps in a blanket of inert gases (e.g., argon, nitrogen) ornon-inert gas such as carbon dioxide to keep the sparged gases in thecontainer head spaces from escaping. Oral liquid formulations preferablyadditionally contain sweeteners and flavorants improve the palatabilityof the formulations.

In one embodiment, as a liquid dosage form, BH4 is stabilized byanti-oxidants and/or by sparging with non-oxidizing, preferablysterilized, gases, such as inert gasses (e.g., nitrogen, argon, helium,etc.) and/or a non-inert gas such as carbon dioxide to remove molecularoxygen from the formulation. The product is preferably filled under ablanket of inert gasses to minimize or prevent molecular oxygen fromredissolving in the formulation. The liquid is filled into a container(e.g., vials, ampoules, etc.) and hermetically sealed to prevent oxygenfrom entering the container. In another embodiment, as a sterile soliddosage form for parenteral administration, a solution of BH4 islyophilized and reconstituted in the clinic prior to administration. Inyet another embodiment, sterile powder drug substance of BH4 is directlypackaged into sterile containers (e.g., vials, bags, bottles orampoules) in a sterile dry powder fill facility. Thus, another aspect ofthe invention is a dry powder formulation of tetrahydrobiopterin (BH4)or a pharmaceutically acceptable salt thereof for constitution into anaqueous solution, including a dry powder mixture of BH4 orpharmaceutically acceptable salt thereof, an antioxidant, and a pHbuffer.

Oral Liquid Formulation Compositions

Oral liquid formulations comprise in addition to the components employedin the general liquid and intravenous formulations, sweeteners andflavoring agents. Sweeteners and flavors are added in quantitiessufficient to yield acceptable sweetness and flavor. Oral liquidformulations contain one or more stabilizers. Optionally, they containantimicrobial preservatives. They are preferentially buffered at low pHe.g., pH 1-4 and the buffering agents are selected to match theflavoring agent thus enhancing the organoleptic properties of the oralliquid formulation. Examples of preferred buffers (acid and conjugatebases) are: citric acid, tartaric acid, malic acid in combination withtheir conjugate bases or salt forms.

Examples of sweeteners include sugars (e.g., sucrose, glucose, sorbitol,mannitol, fructose, etc.), intense non-sugar sweeteners (e.g.,aspartame, acesulfame K, cyclamate, saccharin, sucralose, glycyrrhizin,alitame, neotame, neohesperidine DC, thaumatin, monellin, and the like).

In a further embodiment, for nasal, ophthalmic and otic administrations,BH4 is formulated as discussed for parenteral dosage forms and isoptionally a sterile product. These dosage forms can be provided in akit package presentation with several days of supplies. Each unit withinthe kit can be comprised of one vial or ampoule and one sprayer (fornasal dosage form) or one dropper (in the case of ophthalmic and oticdosage forms). Once the vial or ampoule is opened, the sprayer ordropper is screwed onto the vial or ampoule and the previous cap isdiscarded. The dosage form product is used within a prescribedexpiration period and discarded and a new vial or ampoule is opened foruse. Another embodiment is to fill the solutions in hermetic plasticsingle-use disposable sterile containers produced by aform-fill-and-seal manufacturing process. These packages are opened andthe solutions delivered using the desired route of administration bysqueezing out the liquid contained within them. These dosage forms areadministered once daily and are given via the nostrils (nasal product),or via the eyes (ophthalmic) or droplets are instilled into the auditorycanal (otic product). With respect to medication packaged in form, afill and seal package, the medication is squeezed out onto the route ofadministration.

In a further embodiment, BH4 is administered via buccal and transdermalroutes using formulated strips, patches or films or as topical productsthat placed on the site of delivery. Sublingual tablets are placedbeneath the tongue. These dosage forms are administered once daily andare either attached to the delivery site membrane (buccal andtransdermal route) or placed as a solid or semi-dosage form in thesublingual site. To prevent irritation of the delivery site, a basiccompound such as sodium carbonate or bicarbonate is coated and mixedwith BH4 to prevent interaction with BH4 that would render it unstable.Alternatively the basic compound is added just before use to raise thepH of BH4, which is quite low. Adding the basic excipient at the time ofmanufacturing without coating the alkaline particles to preventinteraction with BH4, will lead to instability of BH4. Anotherembodiment is to coat a core sublingual tablet of BH4 with a coatingsolution containing a basic or alkaline substance. In the sublingualcompartment, the basic compound dissolves first, and interacts with BH4to raise the pH of the medium.

Primary Container Packaging for BH4 Liquid Formulations

The primary packaging containers for BH4 liquid formulations arepreferably impermeable to oxygen, carbon dioxide, nitrogen and inertgases. Following filling of sparged liquid formulations of BH4 into theprimary container, preferably under a blanket of nitrogen, thecontainers are preferably hermetically sealed to keep the sparging gasin the liquid and container headspace and prevent the loss of thesparging gas and ingress of oxygen into the container.

The preferred primary containers are hermetically sealed ampoules aswell as bottles and vials sealed hermetically with metallic cap such asthose employed in sealing soda and beer beverage bottles. During use,the ampoules are cut opened and used within a few hours, e.g., about 12hours. Ampoules can be used for intravenous and sterile products forinjections. Sterile injectable liquids and lyophilized products can alsobe packaged in rubber closure-sealed vials which are secured withcrimped aluminum cap. The antioxidants in the formulations protect theliquid and lyophilized products from the imperceptibly slow loss ofsparged gas or oxygen ingress into the vial for the shelf life of theproduct.

BH4 liquid formulations filled into bottles or vials for oral,ophthalmic or otic use preferably are hermetically secured with abeverage metallic cap or a rubber stopper secured with crimped aluminumseal. The flutes of the bottles or vials can be grooved to accept ascrew cap. When the hermetic seal is removed, it is replaced with ascrew cap with or without a dropper. The presence of antioxidants in theformulation can enable the screw-capped formulation to be stable for usefor at least two weeks, for example, after the hermetic seal is broken.

I. SYNTHESIS OF TETRAHYDROBIOPTERIN

A variety of methods are known in the art for synthesis oftetrahydrobiopterins, precursors, derivatives and analogs. U.S. Pat.Nos. 5,698,408; 2,601,215; 3,505,329; 4,540,783; 4,550,109; 4,587,340;4,595,752; 4,649,197; 4,665,182; 4,701,455; 4,713,454; 4,937,342;5,037,981; 5,198,547; 5,350,851; 5,401,844; 5,698,408, Canadianapplication CA 2420374, European application nos. EP 079 574, EP 191 335and Suntory Japanese patent publications JP 4-082888, JP 59-021685 andJP 9-157270, as well as Sugimoto and Matsuura, Bull. Chem. Soc. Japan,48(12):3767-3768 (1975), Sugimoto and Matsuura, Bull. Chem. Soc. Japan,52(1):181-183 (1979), Matsuura et al., Chem. Lett. (Japan), 735-738(1984), Matsuura et al., Heterocycles, Vol. 23, No. 12, 3115-3120, 1985and Whiteley et al., Anal Biochem. 137(2):394-6 (1984) (eachincorporated herein by reference) each describe methods of makingdihydrobiopterins, BH4 and derivatives thereof that may be used ascompositions for the present invention.

Int'l Publication No. WO2005049614, U.S. Pat. No. 4,540,783, JapanesePatent No. 59-021685, Schircks et al., Helv. Chim. Acta, 60: 211 (1977),Sugimoto et al., Bull. Chem. Soc. Jp, 52(1):181 (1979), Sugimoto et al.,Bull. Chem. Soc. Jp, 48(12):3767 (1975), Visontini et al., Helv. Chim.Acta, 52:1225 (1969), and Matsuura et al., Chem. Lett., p 735 (1984),incorporated herein by reference in their entireties, describe methodsof synthesizing BH4.

II. CRYSTALLINE FORMS OF 6R-TETRAHYDROBIOPTERIN HYDROCHLORIDE SALT

(6R)-L-erythro-tetrahydrobiopterin dihydrochloride exists in differentcrystalline forms, including polymorphic forms and solvates, some ofwhich are more stable than others.

Crystal Polymorph Forms of (6R) L-Tetrahydrobiopterin DihydrochlorideSalt Polymorph Form B

The crystal polymorph that has been found to be the most stable isreferred to herein as “form B,” or alternatively as “polymorph B.”Results obtained during investigation and development of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride development revealedthat there are several known crystalline solids have been prepared, butnone have recognized the polymorphism and its effect on the stability ofthe BH4 crystals.

Polymorph B is a slightly hygroscopic anhydrate with the highestthermodynamic stability above about 20° C. Furthermore, form B can beeasily processed and handled due to its thermal stability, possibilityfor preparation by targeted conditions, its suitable morphology andparticle size. Melting point is near 260° C. (ΔHf >140 J/g), but noclear melting point can be detected due to decomposition prior andduring melting. These outstanding properties render polymorph form Bespecially feasible for pharmaceutical application, which are preparedat elevated temperatures. Polymorph B can be obtained as a fine powderwith a particle size that may range from 0.2 μm to 500 μM.

Form B exhibits an X-ray powder diffraction pattern, expressed ind-values (Å) at: 8.7 (vs), 6.9 (w), 5.90 (vw), 5.63 (m), 5.07 (m), 4.76(m), 4.40 (m), 4.15 (w), 4.00 (s), 3.95 (m), 3.52 (m), 3.44 (w), 3.32(m), 3.23 (s), 3.17 (w), 3.11 (vs), 3.06 (w), 2.99 (w), 2.96 (w), 2.94(m), 2.87 (w), 2.84 (s), 2.82 (m), 2.69 (w), 2.59 (w), 2.44 (w). FIG. 1is a graph of the characteristic X-ray diffraction pattern exhibited byform B of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

As used herein, the following the abbreviations in brackets mean:(vs)=very strong intensity; (s)=strong intensity; (m)=medium intensity;(w)=weak intensity; and (vw)=very weak intensity. A characteristic X-raypowder diffraction pattern is exhibited in FIG. 1.

It has been found that other polymorphs of BH4 have a satisfactorychemical and physical stability for a safe handling during manufactureand formulation as well as providing a high storage stability in itspure form or in formulations. In addition, it has been found that formB, and other polymorphs of BH4 can be prepared in very large quantities(e.g., 100 kilo scale) and stored over an extended period of time.

All crystal forms (polymorphs, hydrates and solvates), inclusive ofcrystal form B, can be used for the preparation of the most stablepolymorph B. Polymorph B may be obtained by phase equilibration ofsuspensions of amorphous or other forms than polymorph form B, such aspolymorph A, in suitable polar and non aqueous solvents. Thus, thepharmaceutical preparations described herein refer to a preparation ofpolymorph form B of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Other forms of BH4 can be converted for form B by dispersing the otherform of BH4 in a solvent at room temperature, stirring the suspension atambient temperatures for a time sufficient to produce polymorph form B,thereafter isolating crystalline form B and removing the solvent fromthe isolated form B. Ambient temperatures, as used herein, meantemperatures in a range from 0° C. to 60° C., preferably 15° C. to 40°C. The applied temperature may be changed during treatment and stirringby decreasing the temperature stepwise or continuously. Suitablesolvents for the conversion of other forms to form B include but are notlimited to, methanol, ethanol, isopropanol, other C3- and C4-alcohols,acetic acid, acetonitrile, tetrahydrofurane, methyl-t-butyl ether,1,4-dioxane, ethyl acetate, isopropyl acetate, other C3-C6-acetates,methyl ethyl ketone and other methyl-C3-C5 alkyl-ketones. The time tocomplete phase equilibration may be up to 30 hours and preferably up to20 hours or less than 20 hours.

Polymorph B may also be obtained by crystallization from solventmixtures containing up to about 5% water, especially from mixtures ofethanol, acetic acid and water. It has been found that polymorph form Bof (6R)-L-erythro-tetrahydrobiopterin dihydrochloride can be prepared bydissolution, optionally at elevated temperatures, preferably of a solidlower energy form than form B or of form B of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in a solvent mixturecomprising ethanol, acetic acid and water, addition of seeds to thesolution, cooling the obtained suspension and isolation of the formedcrystals. Dissolution may be carried out at room temperature or up to70° C., preferably up to 50° C. There may be used the final solventmixture for dissolution or the starting material may be first dissolvedin water and the other solvents may than be added both or one after theother solvent. The composition of the solvent mixture may comprise avolume ratio of water:acetic acid:tetrahydrofuran of 1:3:2 to 1:9:4 andpreferably 1:5:4. The solution is preferably stirred. Cooling may meantemperatures down to −40° C. to 0° C., preferably down to 10° C. to 30°C. Suitable seeds are polymorph form B from another batch or crystalshaving a similar or identical morphology. After isolation, thecrystalline form B can be washed with a non-solvent such as acetone ortetrahydrofurane and dried in usual manner.

Polymorph B may also be obtained by crystallization from aqueoussolutions through the addition of non-solvents such as methanol, ethanoland acetic acid. The crystallization and isolation procedure can beadvantageously carried out at room temperature without cooling thesolution. This process is therefore very suitable to be carried out atan industrial scale.

In one embodiment of the compositions and methods described herein, acomposition including polymorph form B of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is prepared bydissolution of a solid form other than form B or of form B of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in water at ambienttemperatures, adding a non-solvent in an amount sufficient to form asuspension, optionally stirring the suspension for a certain time, andthereafter isolation of the formed crystals. The composition is furthermodified into a pharmaceutical composition as described below.

The concentration of (6R)-L-erythro-tetrahydrobiopterin dihydrochloridein the aqueous solution may be from 10 to 80 percent by weight, morepreferably from 20 to 60 percent by weight, by reference to thesolution. Preferred non-solvents (i.e., solvents useful in preparingsuspensions of BH4) are methanol, ethanol and acetic acid. Thenon-solvent may be added to the aqueous solution. More preferably, theaqueous solution is added to the non-solvent. The stirring time afterformation of the suspension may be up to 30 hours and preferably up to20 hours or less than 20 hours. Isolation by filtration and drying iscarried out in known manner as described above.

Polymorph form B is a very stable crystalline form, that can be easilyfiltered off, dried and ground to particle sizes desired forpharmaceutical formulations. These outstanding properties renderpolymorph form B especially feasible for pharmaceutical application.

Polymorph Form A

It has been found that another crystal polymorph of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form A,” or “polymorph A.”Polymorph A is slightly hygroscopic and adsorbs water to a content ofabout 3 percent by weight, which is continuously released between 50° C.and 200° C., when heated at a rate of 10° C./minute. The polymorph A isa hygroscopic anhydrate, which is a meta-stable form with respect toform B; however, it is stable over several months at ambient conditionsif kept in a tightly sealed container. Form A is especially suitable asintermediate and starting material to produce stable polymorph forms.Polymorph form A can be prepared as a solid powder with desired mediumparticle size range which is typically ranging from 1 μm to about 500μm.

Polymorph A which exhibits a characteristic X-ray powder diffractionpattern with characteristic peaks expressed in d-values (Å) of: 15.5(vs.), 12.0 (m), 6.7 (m), 6.5 (m), 6.3 (w), 6.1 (w), 5.96 (w), 5.49 (m),4.89 (m), 3.79 (m), 3.70 (s), 3.48 (m), 3.45 (m), 3.33 (s), 3.26 (s),3.22 (m), 3.18 (m), 3.08 (m), 3.02 (w), 2.95 (w), 2.87 (m), 2.79 (w),2.70 (w). FIG. 2 is a graph of the characteristic X-ray diffractionpattern exhibited by form A of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

Polymorph A exhibits a characteristic Raman spectra bands, expressed inwave numbers (cm-1) at: 2934 (w), 2880 (w), 1692 (s), 1683 (m), 1577(w), 1462 (m), 1360 (w), 1237 (w), 1108 (w), 1005 (vw), 881 (vw), 813(vw), 717 (m), 687 (m), 673 (m), 659 (m), 550 (w), 530 (w), 492 (m), 371(m), 258 (w), 207 (w), 101 (s), 87 (s) cm-1.

Polymorph form A may be obtained by freeze-drying or water removal ofsolutions of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride inwater. Polymorph form A of (6R)-L-erythro-tetrahydrobiopterindihydrochloride can be prepared by dissolving(6R)-L-erythro-tetrahydrobiopterin dihydrochloride at ambienttemperatures in water, (1) cooling the solution to low temperatures forsolidifying the solution, and removing water under reduced pressure, or(2) removing water from said aqueous solution.

The crystalline form A can be isolated by filtration and then dried toevaporate absorbed water from the product. Drying conditions and methodsare known and drying of the isolated product or water removal pursuantto variant (2) described herein may be carried out in applying elevatedtemperatures, for example up to 80° C., preferably in the range from 30°C. to 80° C., under vacuum or elevated temperatures and vacuum. Prior toisolation of a precipitate obtained in variant (2), the suspension maybe stirred for a certain time for phase equilibration. The concentrationof (6R)-L-erythro-tetrahydrobiopterin dihydrochloride in the aqueoussolution may be from 5 to 40 percent by weight, by reference to thesolution.

A fast cooling is preferred to obtain solid solutions as startingmaterial. A reduced pressure is applied until the solvent is completelyremoved. Freeze drying is a technology well known in the art. The timeto complete solvent removal is dependent on the applied vacuum, whichmay be from 0.01 to 1 mbar, the solvent used and the freezingtemperature.

Polymorph form A is stable at room temperature or below room temperatureunder substantially water free conditions, which is demonstrated withphase equilibration tests of suspensions in tetrahydrofuran ortertiary-butyl methyl ether stirred for five days and 18 hoursrespectively under nitrogen at room temperature. Filtration andair-drying at room temperature yields unchanged polymorph form A.

Polymorph Form F

It has been found that another crystal polymorph of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form F,” or “polymorph F.”Polymorph F is slightly hygroscopic and adsorbs water to a content ofabout 3 percent by weight, which is continuously released between 50° C.and 200° C., when heated at a rate of 10° C./minute. The polymorph F isa meta-stable form and a hygroscopic anhydrate, which is more stablethan form A at ambient lower temperatures and less stable than form B athigher temperatures and form F is especially suitable as intermediateand starting material to produce stable polymorph forms. Polymorph formF can be prepared as a solid powder with desired medium particle sizerange which is typically ranging from 1 μm to about 500 μm.

Polymorph F exhibits a characteristic X-ray powder diffraction patternwith characteristic peaks expressed in d-values (Å) at: 17.1 (vs.), 12.1(w), 8.6 (w), 7.0 (w), 6.5 (w), 6.4 (w), 5.92 (w), 5.72 (w), 5.11 (w),4.92 (m), 4.86 (w), 4.68 (m), 4.41 (w), 4.12 (w), 3.88 (w), 3.83 (w),3.70 (m), 3.64 (w), 3.55 (m), 3.49 (s), 3.46 (vs), 3.39 (s), 3.33 (m),3.31 (m), 3.27 (m), 3.21 (m), 3.19 (m), 3.09 (m), 3.02 (m), and 2.96(m). FIG. 3 is a graph of the characteristic X-ray diffraction patternexhibited by form F of (6R)-L-erythro-tetrahydrobiopterindihydrochloride.

Polymorph F may be obtained by phase equilibration of suspensions ofpolymorph form A in suitable polar and non-aqueous solvents, whichscarcely dissolve said lower energy forms, especially alcohols such asmethanol, ethanol, propanol and isopropanol. Polymorph form F of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride can also be preparedby dispersing particles of solid form A of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in a non-aqueoussolvent that scarcely dissolves said (6R)-L-erythro-tetrahydrobiopterindihydrochloride below room temperature, stiffing the suspension at saidtemperatures for a time sufficient to produce polymorph form F,thereafter isolating crystalline form F and removing the solvent fromthe isolated form F. Removing of solvent and drying may be carried outunder air, dry air or a dry protection gas such as nitrogen or noblegases and at or below room temperature, for example down to 0° C. Thetemperature during phase equilibration is preferably from 5 to 15° C.and most preferably about 10° C.

Polymorph Form J

It has been found that another crystal polymorph of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form J,” or “polymorph J.” Thepolymorph J is slightly hygroscopic and adsorbs water when handled atair humidity. The polymorph J is a meta-stable form and a hygroscopicanhydrate, and it can be transformed back into form E described below,from which it is obtained upon exposure to high relative humidityconditions such as above 75% relative humidity. Form J is especiallysuitable as intermediate and starting material to produce stablepolymorph forms. Polymorph form J can be prepared as a solid powder withdesired medium particle size range which is typically ranging from 1 μmto about 500 μm.

Form J exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 14.6 (m), 6.6 (w),6.4 (w), 5.47 (w), 4.84 (w), 3.29 (vs), and 3.21 (vs). FIG. 4 is a graphof the characteristic X-ray diffraction pattern exhibited by form J of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Polymorph J may be obtained by dehydration of form E at moderatetemperatures under vacuum. In particular, polymorph form J of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride can be prepared bytaking form E and removing the water from form E by treating form E in avacuum drier to obtain form J at moderate temperatures, which may mean atemperature in the range of 25 to 70° C., and most preferably 30 to 50°C.

Polymorph Form K

It has been found that another crystal polymorph of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form K,” or “polymorph K.”Polymorph K is slightly hygroscopic and adsorbs water to a content ofabout 2.0 percent by weight, which is continuously released between 50°C. and 100° C., when heated at a rate of 10° C./minute. The polymorph Kis a meta-stable form and a hygroscopic anhydrate, which is less stablethan form B at higher temperatures and form K is especially suitable asintermediate and starting material to produce stable polymorph forms, inparticular form B. Polymorph form K can be prepared as a solid powderwith desired medium particle size range which is typically ranging from1 μm to about 500 μm.

Form K exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 14.0 (s), 9.4 (w),6.6 (w), 6.4 (w), 6.3 (w), 6.1 (w), 6.0 (w), 5.66 (w), 5.33 (w), 5.13(vw), 4.73 (m), 4.64 (m), 4.48 (w), 4.32 (vw), 4.22 (w), 4.08 (w), 3.88(w), 3.79 (w), 3.54 (m), 3.49 (vs), 3.39 (m), 3.33 (vs), 3.13 (s), 3.10(m), 3.05 (m), 3.01 (m), 2.99 (m), and 2.90 (m). FIG. 5 is a graph ofthe characteristic X-ray diffraction pattern exhibited by form K of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Polymorph K may be obtained by crystallization from mixtures of polarsolvents containing small amounts of water and in the presence of smallamounts of ascorbic acid. Solvents for the solvent mixture may beselected from acetic acid and an alcohol such as methanol, ethanol, n-or isopropanol. In particular, polymorph form K of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride can be prepared bydissolving (6R)-L-erythro-tetrahydrobiopterin dihydrochloride in amixture of acetic acid and an alcohol or tetrahydrofuran containingsmall amounts of water and a small amount of ascorbic acid at elevatedtemperatures, lowering temperature below room temperature to crystallizesaid dihydrochloride, isolating the precipitate and drying the isolatedprecipitate at elevated temperature optionally under vacuum. Suitablealcohols are for example methanol, ethanol, propanol and isopropanol,whereby ethanol is preferred. The ratio of acetic acid to alcohol ortetrahydrofuran may be from 2:1 to 1:2 and preferably about 1:1.Dissolution of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride can becarried out in presence of a higher water content and more of theanti-solvent mixture can be added to obtain complete precipitation. Theamount of water in the final composition may be from 0.5 to 5 percent byweight and the amount of ascorbic acid may be from 0.01 to 0.5 percentby weight, both by reference to the solvent mixture. The temperature fordissolution may be in the range from 30 to 100 and preferably 35 to 70°C. and the drying temperature may be in the range from 30 to 50° C. Theprecipitate may be washed with an alcohol such as ethanol afterisolation, e.g., filtration. The polymorph K can easily be converted inthe most stable form B by phase equilibration in e.g., isopropanol andoptionally seeding with form B crystals at above room temperature suchas temperatures from 30 to 40° C.

Hydrate Forms of (6R) L-Tetrahydrobiopterin Dihydrochloride Salt

As further described below, it has been found that(6R)-L-erythro-tetrahydrobiopterin dihydrochloride exists as a number ofcrystalline hydrate, which shall be described and defined herein asforms C, D, E, H, and O. These hydrate forms are useful as a stable formof BH4 for the pharmaceutical preparations described herein and in thepreparation of compositions including stable crystal polymorphs of BH4.

Hydrate Form C

It has been found that a hydrate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form C,” or “hydrate C.” Thehydrate form C is slightly hygroscopic and has a water content ofapproximately 5.5 percent by weight, which indicates that form C is amonohydrate. The hydrate C has a melting point near 94° C. (ΔH_(f) isabout 31 J/g) and hydrate form C is especially suitable as intermediateand starting material to produce stable polymorphic forms. Polymorphform C can be prepared as a solid powder with desired medium particlesize range which is typically ranging from 1 μm to about 500 μm.

Form C exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 18.2 (m), 15.4 (w),13.9 (vs), 10.4 (w), 9.6 (w), 9.1 (w), 8.8 (m), 8.2 (w), 8.0 (w), 6.8(m), 6.5 (w), 6.05 (m), 5.77 (w), 5.64 (w), 5.44 (w), 5.19 (w), 4.89(w), 4.76 (w), 4.70 (w), 4.41 (w), 4.25 (m), 4.00 (m), 3.88 (m), 3.80(m), 3.59 (s), 3.50 (m), 3.44 (m), 3.37 (m), 3.26 (s), 3.19 (vs), 3.17(s), 3.11 (m), 3.06 (m), 3.02 (m), 2.97 (vs), 2.93 (m), 2.89 (m), 2.83(m), and 2.43 (m). FIG. 6 is a graph of the characteristic X-raydiffraction pattern exhibited by hydrate form C of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Hydrate form C may be obtained by phase equilibration at ambienttemperatures of a polymorph form such as polymorph B suspension in anon-solvent, which contains water in an amount of preferably about 5percent by weight, by reference to the solvent. Hydrate form C of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride cab be prepared bysuspending (6R)-L-erythro-tetrahydrobiopterin dihydrochloride in anon-solvent such as, heptane, C1-C4-alcohols such as methanol, ethanol,1- or 2-propanol, acetates, such as ethyl acetate, acetonitrile, aceticacid or ethers such as terahydrofuran, dioxane, tertiary-butyl methylether, or binary or ternary mixtures of such non-solvents, to whichsufficient water is added to form a monohydrate, and stirring thesuspension at or below ambient temperatures (e.g., 0 to 30° C.) for atime sufficient to form a monohydrate. Sufficient water may mean from 1to 10 and preferably from 3 to 8 percent by weight of water, byreference to the amount of solvent. The solids may be filtered off anddried in air at about room temperature. The solid can absorb some waterand therefore possess a higher water content than the theoretical valueof 5.5 percent by weight. Hydrate form C is unstable with respect toforms D and B, and easily converted to polymorph form B at temperaturesof about 40° C. in air and lower relative humidity. Form C can betransformed into the more stable hydrate D by suspension equilibrationat room temperature.

Hydrate Form D

It has been found that another hydrate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form D,” or “hydrate D.” Thehydrate form D is slightly hygroscopic and may have a water content ofapproximately 5.0 to 7.0 percent by weight, which suggests that form Dis a monohydrate. The hydrate D has a melting point near 153° C. (ΔH_(f)is about 111 J/g) and is of much higher stability than form C and iseven stable when exposed to air humidity at ambient temperature. Hydrateform D can therefore either be used to prepare formulations or asintermediate and starting material to produce stable polymorph forms.Polymorph form D can be prepared as a solid powder with desired mediumparticle size range which is typically ranging from 1 μm to about 500μm.

Form D exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 8.6 (s), 6.8 (w),5.56 (m), 4.99 (m), 4.67 (s), 4.32 (m), 3.93 (vs), 3.88 (w), 3.64 (w),3.41 (w), 3.25 (w), 3.17 (m), 3.05 (s), 2.94 (w), 2.92 (w), 2.88 (m),2.85 (w), 2.80 (w), 2.79 (m), 2.68 (w), 2.65 (w), 2.52 (vw), 2.35 (w),2.34 (w), 2.30 (w), and 2.29 (w). FIG. 7 is a graph of thecharacteristic X-ray diffraction pattern exhibited by hydrate form D of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Hydrate form D may be obtained by adding at about room temperatureconcentrated aqueous solutions of (6R)-L-erythro-tetrahydrobiopterindihydrochloride to an excess of a non-solvent such as hexane, heptane,dichloromethane, 1- or 2-propanol, acetone, ethyl acetate, acetonitrile,acetic acid or ethers such as terahydrofuran, dioxane, tertiary-butylmethyl ether, or mixtures of such non-solvents, and stirring thesuspension at ambient temperatures. The crystalline solid can befiltered off and then dried under dry nitrogen at ambient temperatures.A preferred non-solvent is isopropanol. The addition of the aqueoussolution may carried out drop-wise to avoid a sudden precipitation.Hydrate form D of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride canbe prepared by adding at about room temperature a concentrated aqueoussolutions of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride to anexcess of a non-solvent and stirring the suspension at ambienttemperatures. Excess of non-solvent may mean a ratio of aqueous to thenon-solvent from 1:10 to 1:1000. Form D contains a small excess ofwater, related to the monohydrate, and it is believed that it isabsorbed water due to the slightly hygroscopic nature of thiscrystalline hydrate. Hydrate form D is deemed to be the most stable oneunder the known hydrates at ambient temperatures and a relative humidityof less than 70%. Hydrate form D may be used for formulations preparedunder conditions, where this hydrate is stable. Ambient temperature maymean 20 to 30° C.

Hydrate Form E

It has been found that another hydrate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form E,” or “hydrate E.” Thehydrate form E has a water content of approximately 10 to 14 percent byweight, which suggests that form E is a dihydrate. The hydrate E isformed at temperatures below room temperature. Hydrate form E isespecially suitable as intermediate and starting material to producestable polymorph forms. It is especially suitable to produce thewater-free form J upon drying under nitrogen or optionally under vacuum.Form E is non-hygroscopic and stable under rather high relativehumidities, i.e., at relative humidities above about 60% and up to about85%. Polymorph form E can be prepared as a solid powder with desiredmedium particle size range which is typically ranging from 1 μm to about500 μm.

Form E exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 15.4 (s), 6.6 (w),6.5 (w), 5.95 (vw), 5.61 (vw), 5.48 (w), 5.24 (w), 4.87 (w), 4.50 (vw),4.27 (w), 3.94 (w), 3.78 (w), 3.69 (m), 3.60 (w), 3.33 (s), 3.26 (vs),3.16 (w), 3.08 (m), 2.98 (w), 2.95 (m), 2.91 (w), 2.87 (m), 2.79 (w),2.74 (w), 2.69 (w), and 2.62 (w). FIG. 8 is a graph of thecharacteristic X-ray diffraction pattern exhibited by hydrate form E of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Hydrate form E may be obtained by adding concentrated aqueous solutionsof (6R)-L-erythro-tetrahydrobiopterin dihydrochloride to an excess of anon-solvent cooled to temperatures from about 10 to −10° C. andpreferably between 0 to 10° C. and stirring the suspension at saidtemperatures. The crystalline solid can be filtered off and then driedunder dry nitrogen at ambient temperatures. Non-solvents are for examplesuch as hexane, heptane, dichloromethane, 1- or 2-propanol, acetone,ethyl acetate, acetonitrile, acetic acid or ethers such asterahydrofuran, dioxane, tertiary-butyl methyl ether, or mixtures ofsuch non-solvents. A preferred non-solvent is isopropanol. The additionof the aqueous solution may carried out drop-wise to avoid a suddenprecipitation. Hydrate form E of (6R)-L-erythro-tetrahydrobiopterindihydrochloride can be prepared by adding a concentrated aqueoussolutions of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride to anexcess of a non-solvent, which is cooled to temperatures from about 10to −10° C., and stirring the suspension at ambient temperatures. Excessof non-solvent may mean a ratio of aqueous to the non-solvent from 1:10to 1:1000. A preferred non-solvent is tetrahydrofuran. Anotherpreparation process comprises exposing polymorph form B to an airatmosphere with a relative humidity of 70 to 90%, preferably about 80%.Hydrate form E is deemed to be a dihydrate, whereby some additionalwater may be absorbed. Polymorph form E can be transformed intopolymorph J upon drying under vacuum at moderate temperatures, which maymean between 20° C. and 50° C. at pressures between 0 and 100 mbar. FormE is especially suitable for formulations in semi solid forms because ofits stability at high relative humidities.

Hydrate Form H

It has been found that another hydrate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form H,” or “hydrate H.” Thehydrate form H has a water content of approximately 5.0 to 7.0 percentby weight, which suggests that form H is a hygroscopic monohydrate. Thehydrate form H is formed at temperatures below room temperature. Hydrateform H is especially suitable as intermediate and starting material toproduce stable polymorph forms. Polymorph form H can be prepared as asolid powder with desired medium particle size range which is typicallyranging from 1 μm to about 500 μm.

Form H exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 8.6 15.8 (vs), 10.3(w), 8.0 (w), 6.6 (w), 6.07 (w), 4.81 (w), 4.30 (w), 3.87 (m), 3.60 (m),3.27 (m), 3.21 (m), 3.13 (w), 3.05 (w), 2.96 (m), 2.89 (m), 2.82 (w),and 2.67 (m). FIG. 9 is a graph of the characteristic X-ray diffractionpattern exhibited by hydrate form H of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Hydrate form H may be obtained by dissolving at ambient temperatures(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in a mixture ofacetic acid and water, adding then a non-solvent to precipitate acrystalline solid, cooling the obtained suspension and stirring thecooled suspension for a certain time. The crystalline solid is filteredoff and then dried under vacuum at ambient temperatures. Non-solventsare for example such as hexane, heptane, dichloromethane, 1- or2-propanol, acetone, ethyl acetate, acetonitril, acetic acid or etherssuch as terahydrofuran, dioxane, tertiary-butyl methyl ether, ormixtures of such non-solvents. A preferred non-solvent istetrahydrofuran. Hydrate form H of (6R)-L-erythro-tetrahydrobiopterindihydrochloride can be by prepared by dissolving at ambient temperatures(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in a mixture ofacetic acid and a less amount than that of acetic acid of water, addinga non-solvent and cooling the obtained suspension to temperatures in therange of −10 to 10° C., and preferably −5 to 5° C., and stirring thesuspension at said temperature for a certain time. Certain time may mean1 to 20 hours. The weight ratio of acetic acid to water may be from 2:1to 25:1 and preferably 5:1 to 15:1. The weight ratio of aceticacid/water to the non-solvent may be from 1:2 to 1:5. Hydrate form Hseems to be a monohydrate with a slight excess of water absorbed due tothe hygroscopic nature.

Hydrate Form O

It has been found that another hydrate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form O,” or “hydrate O.” Thehydrate form O is formed at temperatures near room temperature. Hydrateform O is especially suitable as intermediate and starting material toproduce stable polymorph forms. Polymorph form O can be prepared as asolid powder with desired medium particle size range which is typicallyranging from 1 μm to about 500 μm.

Form O exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 15.9 (w), 14.0 (w),12.0 (w), 8.8 (m), 7.0 (w), 6.5 (w), 6.3 (m), 6.00 (w), 5.75 (w), 5.65(m), 5.06 (m), 4.98 (m), 4.92 (m), 4.84 (w), 4.77 (w), 4.42 (w), 4.33(w), 4.00 (m), 3.88 (m), 3.78 (w), 3.69 (s), 3.64 (s), 3.52 (vs), 3.49(s), 3.46 (s), 3.42 (s), 3.32 (m), 3.27 (m), 3.23 (s), 3.18 (s), 3.15(vs), 3.12 (m), 3.04 (vs), 2.95 (m), 2.81 (s), 2.72 (m), 2.67 (m), and2.61 (m). FIG. 10 is a graph of the characteristic X-ray diffractionpattern exhibited by hydrate form O of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Hydrate form O can be prepared by exposure of polymorphic form F to anitrogen atmosphere containing water vapor with a resulting relativehumidity of about 52% for about 24 hours. The fact that form F, which isa slightly hygroscopic anhydrate, can be used to prepare form O under52% relative humidity suggests that form O is a hydrate, which is morestable than form F under ambient temperature and humidity conditions.

Solvate Forms of (6R) L-Tetrahydrobiopterin Dihydrochloride Salt

As further described below, it has been found that(6R)-L-erythro-tetrahydrobiopterin dihydrochloride exists as a number ofcrystalline solvate forms, which shall be described and defined hereinas forms G, I, L, M, and N. These solvate forms are useful as a stableform of BH4 for the pharmaceutical preparations described herein and inthe preparation of compositions including stable crystal polymorphs ofBH4.

Solvate Form G

It has been found that an ethanol solvate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form G,” or “hydrate G.” Theethanol solvate form G has a ethanol content of approximately 8.0 to12.5 percent by weight, which suggests that form G is a hygroscopic monoethanol solvate. The solvate form G is formed at temperatures below roomtemperature. Form G is especially suitable as intermediate and startingmaterial to produce stable polymorph forms. Polymorph form G can beprepared as a solid powder with a desired medium particle size rangewhich is typically ranging from 1 μm to about 500 μm.

Form G exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 14.5 (vs), 10.9 (w),9.8 (w), 7.0 (w), 6.3 (w), 5.74 (w), 5.24 (vw), 5.04 (vw), 4.79 (w),4.41 (w), 4.02 (w), 3.86 (w), 3.77 (w), 3.69 (w), 3.63 (m), 3.57 (m),3.49 (m), 3.41 (m), 3.26 (m), 3.17 (m), 3.07 (m), 2.97 (m), 2.95 (m),2.87 (w), and 2.61 (w). FIG. 11 is a graph of the characteristic X-raydiffraction pattern exhibited by solvate form G of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Ethanol solvate form G may be obtained by crystallization ofL-erythro-tetrahydrobiopterin dihydrochloride dissolved in water andadding a large excess of ethanol, stirring the obtained suspension at orbelow ambient temperatures and drying the isolated solid under air ornitrogen at about room temperature. Here, a large excess of ethanolmeans a resulting mixture of ethanol and water with less than 10% water,preferably about 3 to 6%. Ethanolate form G of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride can be prepared bydissolving at about room temperature to temperatures of 75° C.(6R)-L-erythro-tetrahydrobiopterin dihydrochloride in water or in amixture of water and ethanol, cooling a heated solution to roomtemperature and down to 5 to 10° C., adding optionally ethanol tocomplete precipitation, stirring the obtained suspension at temperaturesof 20 to 5° C., filtering off the white, crystalline solid and dryingthe solid under air or a protection gas such as nitrogen at temperaturesabout room temperature. The process may be carried out in a firstvariant in dissolving (6R)-L-erythro-tetrahydrobiopterin dihydrochlorideat about room temperature in a lower amount of water and then adding anexcess of ethanol and then stiffing the obtained suspension for a timesufficient for phase equilibration. In a second variant,(6R)-L-erythro-tetrahydrobiopterin dihydrochloride may be suspended inethanol, optionally adding a lower amount of water, and heating thesuspension and dissolute (6R)-L-erythro-tetrahydrobiopterindihydrochloride, cooling down the solution to temperatures of about 5 to15° C., adding additional ethanol to the suspension and then stirringthe obtained suspension for a time sufficient for phase equilibration.

Solvate Form I

It has been found that an acetic acid solvate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form I,” or “hydrate I.” Theacetic acid solvate form I has an acetic acid content of approximately12.7 percent by weight, which suggests that form I is a hygroscopicacetic acid mono solvate. The solvate form I is formed at temperaturesbelow room temperature. Acetic acid solvate form I is especiallysuitable as intermediate and starting material to produce stablepolymorph forms. Polymorph form I can be prepared as a solid powder withdesired medium particle size range which is typically ranging from 1 μmto about 500 μm.

Form I exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 14.5 (m), 14.0 (w),11.0 (w), 7.0 (vw), 6.9 (vw), 6.2 (vw), 5.30 (w), 4.79 (w), 4.44 (w),4.29 (w), 4.20 (vw), 4.02 (w), 3.84 (w), 3.80 (w), 3.67 (vs), 3.61 (m),3.56 (w), 3.44 (m), 3.27 (w), 3.19 (w), 3.11 (s), 3.00 (m), 2.94 (w),2.87 (w), and 2.80 (w). FIG. 12 is a graph of the characteristic X-raydiffraction pattern exhibited by solvate form I of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Acetic acid solvate form I may be obtained by dissolution ofL-erythro-tetrahydrobiopterin dihydrochloride in a mixture of aceticacid and water at elevated temperature, adding further acetic acid tothe solution, cooling down to a temperature of about 10° C., thenwarming up the formed suspension to about 15° C., and then stifling theobtained suspension for a time sufficient for phase equilibration, whichmay last up to 3 days. The crystalline solid is then filtered off anddried under air or a protection gas such as nitrogen at temperaturesabout room temperature.

Solvate Form L

It has been found that a mixed ethanol solvate/hydrate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form L,” or “hydrate L.” Form Lmay contain 4% but up to 13% ethanol and 0% to about 6% of water. Form Lmay be transformed into form G when treated in ethanol at temperaturesfrom about 0° C. to 20° C. In addition form L may be transformed intoform B when treated in an organic solvent at ambient temperatures (10°C. to 60° C.). Polymorph form L can be prepared as a solid powder withdesired medium particle size range which is typically ranging from 1 μmto about 500 μm.

Form L exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 14.1 (vs), 10.4 (w),9.5 (w), 9.0 (vw), 6.9 (w), 6.5 (w), 6.1 (w), 5.75 (w), 5.61 (w), 5.08(w), 4.71 (w), 3.86 (w), 3.78 (w), 3.46 (m), 3.36 (m), 3.06 (w), 2.90(w), and 2.82 (w). FIG. 13 is a graph of the characteristic X-raydiffraction pattern exhibited by solvate form L of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Form L may be obtained by suspending hydrate form E at room temperaturein ethanol and stirring the suspension at temperatures from 0 to 10° C.,preferably about 5° C., for a time sufficient for phase equilibration,which may be 10 to 20 hours. The crystalline solid is then filtered offand dried preferably under reduced pressure at 30° C. or under nitrogen.Analysis by TG-FTIR suggests that form L may contain variable amounts ofethanol and water, i.e., it can exist as an polymorph (anhydrate), as amixed ethanol solvate/hydrate, or even as a hydrate.

Solvate Form M

It has been found that an ethanol solvate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form M,” or “hydrate M.” Form Mmay contain 4% but up to 13% ethanol and 0% to about 6% of water, whichsuggests that form M is a slightly hygroscopic ethanol solvate. Thesolvate form M is formed at room temperature. Form M is especiallysuitable as intermediate and starting material to produce stablepolymorph forms, since form M can be transformed into form G whentreated in ethanol at temperatures between about −10° to 15° C., andinto form B when treated in organic solvents such as ethanol, C3 and C4alcohols, or cyclic ethers such as THF and dioxane. Polymorph form M canbe prepared as a solid powder with desired medium particle size rangewhich is typically ranging from 1 μm to about 500 μm.

Form M exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 18.9 (s), 6.4 (m),6.06 (w), 5.66 (w), 5.28 (w), 4.50 (w), 4.23 (w), and 3.22 (vs). FIG. 14is a graph of the characteristic X-ray diffraction pattern exhibited bysolvate form M of (6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

Ethanol solvate form M may be obtained by dissolution ofL-erythro-tetrahydrobiopterin dihydrochloride in ethanol and evaporationof the solution under nitrogen at ambient temperature, i.e., between 10°C. and 40° C. Form M may also be obtained by drying of form G under aslight flow of dry nitrogen at a rate of about 20 to 100 ml/min.Depending on the extent of drying under nitrogen, the remaining amountof ethanol may be variable, i.e., from about 3% to 13%.

Solvate Form N

It has been found that another solvate crystal form of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride is a stable preferredform of BH4 for use in a pharmaceutical preparation described herein,which shall be referred to herein as “form N,” or “hydrate N.” Form Nmay contain in total up to 10% of isopropanol and water, which suggeststhat form N is a slightly hygroscopic isopropanol solvate. Form N may beobtained through washing of form D with isopropanol and subsequentdrying in vacuum at about 30° C. Form N is especially suitable asintermediate and starting material to produce stable polymorph forms.Polymorph form N can be prepared as a solid powder with desired mediumparticle size range which is typically ranging from 1 μm to about 500μm.

Form N exhibits a characteristic X-ray powder diffraction pattern withcharacteristic peaks expressed in d-values (Å) at: 19.5 (m), 9.9 (w),6.7 (w), 5.15 (w), 4.83 (w), 3.91 (w), 3.56 (m), 3.33 (vs), 3.15 (w),2.89 (w), 2.81 (w), 2.56 (w), and 2.36 (w). FIG. 15 is a graph of thecharacteristic X-ray diffraction pattern exhibited by solvate form N of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride.

The isopropanol form N may be obtained by dissolution ofL-erythro-tetrahydrobiopterin dihydrochloride in 4.0 ml of a mixture ofisopropanol and water (mixing volume ratio for example 4:1). To thissolution is slowly added isopropanol (IPA, for example about 4.0 ml) andthe resulting suspension is cooled to 0° C. and stirred for severalhours (e.g., about 10 to 18 hours) at this temperature. The suspensionis filtered and the solid residue washed with isopropanol at roomtemperature. The obtained crystalline material is then dried at ambienttemperature (e.g., about 20 to 30° C.) and reduced pressure (about 2 to10 mbar) for several hours (e.g., about 5 to 20 hours). TG-FTIR shows aweight loss of 9.0% between 25 to 200° C., which is attributed to bothisopropanol and water. This result suggests that form N can exist eitherin form of an isopropanol solvate, or in form of mixed isopropanolsolvate/hydrate, or as an non-solvated form containing a small amount ofwater.

For the preparation of the polymorph forms, there may be usedcrystallization techniques well known in the art, such as stirring of asuspension (phase equilibration in), precipitation, re-crystallization,evaporation, solvent like water sorption methods or decomposition ofsolvates. Diluted, saturated or super-saturated solutions may be usedfor crystallization, with or without seeding with suitable nucleatingagents. Temperatures up to 100° C. may be applied to form solutions.Cooling to initiate crystallization and precipitation down to −100° C.and preferably down to −30° C. may be applied. Meta-stable polymorphs orpseudo-polymorphic forms can be used to prepare solutions or suspensionsfor the preparation of more stable forms and to achieve higherconcentrations in the solutions.

It was surprisingly found that hydrate form D is the most stable formunder the hydrates and forms B and D are especially suitable to be usedin pharmaceutical formulations. Forms B and D presents some advantageslike an aimed manufacture, good handling due to convenient crystal sizeand morphology, very good stability under production conditions ofvarious types of formulation, storage stability, higher solubility, andhigh bioavailability. Accordingly, one embodiment of the compositionsand methods disclosed herein is pharmaceutical composition includingpolymorph form B and/or hydrate form D of(6R)-L-erythro-tetrahydrobiopterin dihydrochloride and apharmaceutically acceptable carrier or diluent.

III. PHARMACEUTICAL FORMULATIONS

The formulations described herein are preferably administered as oralformulations. Oral formulations are preferably solid formulations suchas capsules, tablets, pills and troches, or liquid formulations such asaqueous suspensions, elixirs and syrups. The various form of BH4described herein can be directly used as powder (micronized particles),granules, suspensions or solutions, or it may be combined together withother pharmaceutically acceptable ingredients in admixing the componentsand optionally finely divide them, and then filling capsules, composedfor example from hard or soft gelatin, compressing tablets, pills ortroches, or suspend or dissolve them in carriers for suspensions,elixirs and syrups. Coatings may be applied after compression to formpills.

Pharmaceutically acceptable ingredients are well known for the varioustypes of formulation and may be, for example, binders such as natural orsynthetic polymers, excipients, lubricants, surfactants, sweetening andflavouring agents, coating materials, preservatives, dyes, thickeners,adjuvants, antimicrobial agents, antioxidants and carriers for thevarious formulation types. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that are approved by the U.S. Food and Drug Administrationor a corresponding foreign regulatory agency for administration tohumans. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the therapeutic compositions, its use intherapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The initial amount of (6R)-L-erythro-tetrahydrobiopterin used to preparethe formulation may be, for example, in the range of about 30 wt % toabout 40 wt % of the formulation, or in the range of about 32 wt % toabout 35 wt %, or at about 33 wt %. Specific amounts of BH4 in aformulation contemplated herein include 80 mg, 100 mg, 200 mg, 300 mg,400 mg, and 500 mg.

Binders assist in maintaining a solid formulation. In some cases,anhydrous binders are used to preserve the anhydrous state of polymorphforms. In some cases, the binder may act as a drying agent. Exemplarybinders include anhydrous dibasic calcium phosphate and its monohydrate.Other nonlimiting examples of binders useful in a composition describedherein include gum tragacanth, acacia, starch, gelatine, and biologicaldegradable polymers such as homo- or co-polyesters of dicarboxylicacids, alkylene glycols, polyalkylene glycols and/or aliphatic hydroxylcarboxylic acids; homo- or co-polyamides of dicarboxylic acids, alkylenediamines, and/or aliphatic amino carboxylic acids; correspondingpolyester-polyamide-co-polymers, polyanhydrides, polyorthoesters,polyphosphazene and polycarbonates. The biological degradable polymersmay be linear, branched or crosslinked. Specific examples arepoly-glycolic acid, poly-lactic acid, and poly-d,l-lactide/glycolide.Other examples for polymers are water-soluble polymers such aspolyoxaalkylenes (polyoxaethylene, polyoxapropylene and mixed polymersthereof, poly-acrylamides and hydroxylalkylated polyacrylamides,poly-maleic acid and esters or -amides thereof, poly-acrylic acid andesters or -amides thereof, poly-vinylalcohol und esters or -ethersthereof, poly-vinylimidazole, poly-vinylpyrrolidon, und natural polymerslike chitosan.

Disintegration agents assist in rapid disintegration of solidformulations by absorbing water and expanding. Exemplary disintegrationagents include polyvinylpyrrolidone (PVP, e.g. sold under the namePOVIDONE), a cross-linked form of povidone (CPVP, e.g. sold under thename CROSPOVIDONE), a cross-linked form of sodium carboxymethylcellulose(NaCMC, e.g. sold under the name AC-DI-SOL), other modified celluloses,and modified starch. Tablets formulated with CPVP exhibited much morerapid disintegration than tablets formulated with PVP.

Antioxidants may be included and help stabilize the tetrahydrobiopterinproduct, especially after dissolution. Low pH aqueous solutions of APIare more stable than are solutions at neutral or high pH. Antioxidantsare included in a formulation described herein to prevent deteriorationfrom oxidation. Antioxidants can generally be classified into 3 groups.

The first group is known as true antioxidants, and inhibit oxidation byreacting with free radicals blocking the chain reaction. Examplesinclude phenolic antioxidants, including butylated hydroxyanisol (BHA),butylated hydroxytoluene (BHT), tert-butyl-hydroquinone (TBHQ),4-hydroxymethyl-2,6-di-tert-butylphenol (HMBP), and2,4,5-trihydroxybutyrophenone (THBP); alkygallates, including propylgallate; gallic acid; nordihydroguaiaretic acid; and tocopherols,including alpha-tocopherol.

The second group, consisting of reducing agents, have lower redoxpotentials than the drug which they are intended to protect, and aretherefore more readily oxidized. Reducing agents may act also byreacting with free radicals. Examples include ascorbic acid,thioglycolic acid (TGA), ascorbyl palmitate, sulfites, includingpotassium and sodium salts of sulphurous acid (e.g., potassium sulfite,sodium sulfite, sodium metabisulphite, and sodium bisulfite), andthioglycerol.

The third group consists of antioxidant synergists which usually have amodest antioxidant effect themselves but probably enhance the action ofantioxidants in the first or second group by reacting with heavy metalions which catalyze oxidation. Examples of such antioxidant synergistsand chelating agents include citric acid, malic acid, editic acid andits salts, lecithin, and tartaric acid.

Exemplary acidic antioxidants include ascorbic acid, fatty acid estersof ascorbic acid such as ascorbyl palmitate and ascorbyl stearate, andsalts of ascorbic acid such as sodium, calcium, or potassium ascorbate.Non-acidic antioxidants may also be used in the stable tabletformulations. Nonlimiting examples of non-acidic antioxidants includebeta-carotene, alpha-tocopherol. Acidic additives may be added toenhance stability of the tablet formulation, including citric acid ormalic acid. Small molecule anti-oxidants include but are not limited tothiols, e.g., cysteine, N-acetyl cysteine, gluthatione, etc., orthiolated polymers (polymer-SH), e.g., polycarbophil-cysteine,polymethacrylic-SH, carboxy methylcellulose-cysteine, etc. or smallmolecule anti-oxidants such as ascorbic acid, methionine, ascorbylpalmitate, etc. These anti-oxidants confer stability on the dosage formduring transit through the GIT, particularly as the pH of the GITincreases with distance from the stomach.

In one embodiment, a combination of at least two reducing agentantioxidants is preferred. In another embodiment, a combination of atleast two reducing agent antioxidants together with an acid antioxidantsynergist and/or chelating agent is preferred.

Lubricants improve stability, hardness and uniformity of solidformulations. Exemplary lubricants include stearyl fumarate andmagnesium stearate. Other nonlimiting examples of lubricants includenatural or synthetic oils, fats, waxes, or fatty acid salts such asmagnesium stearate.

Optionally the stable formulations of the invention can also compriseother excipients such as mannitol, hydroxylpropyl cellulose,microcrystalline cellulose, or other non-reducing sugars such assucrose, trehalose, melezitose, planteose, and raffinose. Reducingsugars may react with BH4. Other nonlimiting examples of excipientsuseful in a composition described herein include phosphates such asdicalcium phosphate.

Surfactants for use in a composition described herein can be anionic,anionic, amphoteric or neutral. Nonlimiting examples of surfactantsuseful in a composition described herein include lecithin,phospholipids, octyl sulfate, decyl sulfate, dodecyl sulfate, tetradecylsulfate, hexadecyl sulfate and octadecyl sulfate, Na oleate or Nacaprate, 1-acylaminoethane-2-sulfonic acids, such as1-octanoylaminoethane-2-sulfonic acid, 1-decanoylaminoethane-2-sulfonicacid, 1-dodecanoylaminoethane-2-sulfonic acid,1-tetradecanoylaminoethane-2-sulfonic acid,1-hexadecanoylaminoethane-2-sulfonic acid, and1-octadecanoylaminoethane-2-sulfonic acid, and taurocholic acid andtaurodeoxycholic acid, bile acids and their salts, such as cholic acid,deoxycholic acid and sodium glycocholates, sodium caprate or sodiumlaurate, sodium oleate, sodium lauryl sulphate, sodium cetyl sulphate,sulfated castor oil and sodium dioctylsulfosuccinate,cocamidopropylbetaine and laurylbetaine, fatty alcohols, cholesterols,glycerol mono- or -distearate, glycerol mono- or -dioleate and glycerolmono- or -dipalmitate, and polyoxyethylene stearate.

Nonlimiting examples of sweetening agents useful in a compositiondescribed herein include sucrose, fructose, lactose or aspartame.Nonlimiting examples of flavoring agents for use in a compositiondescribed herein include peppermint, oil of wintergreen or fruit flavorssuch as cherry or orange flavor. Nonlimiting examples of coatingmaterials for use in a composition described herein include gelatin,wax, shellac, sugar or other biological degradable polymers. Nonlimitingexamples of preservatives for use in a composition described hereininclude methyl or propylparabens, sorbic acid, chlorobutanol, phenol andthimerosal.

The BH4 form may also be formulated as effervescent tablet or powder,which disintegrate in an aqueous environment to provide a drinkingsolution. Slow release formulations may also be prepared in order toachieve a controlled release of the active agent in contact with thebody fluids in the gastro intestinal tract, and to provide a substantialconstant and effective level of the active agent in the blood plasma.The crystal form may be embedded for this purpose in a polymer matrix ofa biological degradable polymer, a water-soluble polymer or a mixture ofboth, and optionally suitable surfactants. Embedding can mean in thiscontext the incorporation of micro-particles in a matrix of polymers.Controlled release formulations are also obtained through encapsulationof dispersed micro-particles or emulsified micro-droplets via knowndispersion or emulsion coating technologies.

The BH4 used in a composition described herein is preferably formulatedas a dihydrochloride salt, however, it is contemplated that other saltforms of BH4 possess the desired biological activity, and consequently,other salt forms of BH4 can be used. Specifically, BH4 salts withinorganic or organic acids are preferred. Nonlimiting examples ofalternative BH4 salts forms includes BH4 salts of acetic acid, citricacid, oxalic acid, tartaric acid, fumaric acid, and mandelic acid.

Pharmaceutically acceptable base addition salts may be formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Pharmaceutically acceptable salts of compounds may also beprepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.Examples of metals used as cations are sodium, potassium, magnesium,ammonium, calcium, or ferric, and the like. Examples of suitable aminesinclude isopropylamine, trimethylamine, histidine, N,N′dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N methylglucamine, and procaine.

Pharmaceutically acceptable acid addition salts include inorganic ororganic acid salts. Examples of suitable acid salts include thehydrochlorides, acetates, citrates, salicylates, nitrates, phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include, for example, acetic, citric, oxalic,tartaric, or mandelic acids, hydrochloric acid, hydrobromic acid,sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic,sulfo or phospho acids or N substituted sulfamic acids, for exampleacetic acid, propionic acid, glycolic acid, succinic acid, maleic acid,hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid,tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid,glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, salicylic acid, 4 aminosalicylic acid, 2 phenoxybenzoic acid, 2acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid;and with amino acids, such as the 20 alpha amino acids involved in thesynthesis of proteins in nature, for example glutamic acid or asparticacid, and also with phenylacetic acid, methanesulfonic acid,ethanesulfonic acid, 2 hydroxyethanesulfonic acid, ethane 1,2 disulfonicacid, benzenesulfonic acid, 4 methylbenzenesulfoc acid, naphthalene 2sulfonic acid, naphthalene 1,5 disulfonic acid, 2 or 3 phosphoglycerate,glucose 6 phosphate, N cyclohexylsulfamic acid (with the formation ofcyclamates), or with other acid organic compounds, such as ascorbicacid.

Exemplary stable oral formulations contain one or more of the followingadditional ingredients that improve the stability or othercharacteristics of the formulation: binder, disintegration agent, acidicantioxidant, or lubricant or combinations thereof. Exemplary stabletablet formulations include a binder and disintegration agent,optionally with an acidic antioxidant, and optionally further includinga lubricant. Exemplary concentrations of binder are between about 1 wt %to about 5 wt %, or between about 1.5 and 3 wt %; an exemplary weightratio of binder to BH4 is in the range of about 1:10 to about 1:20.Exemplary concentrations of disintegration agent are between about 1 wt% to about 20 wt %; an exemplary weight ratio of disintegration agent toBH4 is in the range of about 1:5 to about 1:10. Exemplary concentrationsof antioxidant are between about 1 wt % and about 3 wt %; an exemplaryweight ratio of antioxidant to BH4 is in the range of about 1:5 to 1:30.In one example, ascorbic acid is the antioxidant and is used at a ratioto BH4 of less than 1:1, e.g. 1:2 or less, or 1:10 or less. Exemplaryconcentrations of lubricant in a stable tablet formulation of thepresent invention are between about 0.1 wt % and about 2 wt %; anexemplary weight ratio of lubricant to BH4 is in the range of about 1:25to 1:65.

The stable solid formulation may optionally include other therapeuticagents suitable for the condition to be treated, e.g. folates, includingfolate precursors, folic acids, or folate derivatives; and/or arginine;and/or vitamins, such as vitamin C and/or vitamin B2 (riboflavin) and/orvitamin B12; and/or neurotransmitter precursors such as L-dopa orcarbidopa; and/or 5-hydroxytryptophan.

Exemplary folates, including folate precursors, folic acids, or folatederivatives, are disclosed in U.S. Pat. Nos. 6,011,040 and 6,544,994,both of which are incorporated herein by reference, and include folicacid (pteroylmonoglutamate), dihydrofolic acid, tetrahydrofolic acid,5-methyltetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid,5,10-methenyltetrahydrofolic acid, 5,10-formiminotetrahydrofolic acid,5-formyltetrahydrofolic acid (leucovorin), 10-formyltetrahydrofolicacid, 10-methyltetrahydrofolic acid, one or more of thefolylpolyglutamates, compounds in which the pyrazine ring of the pterinmoiety of folic acid or of the folylpolyglutamates is reduced to givedihydrofolates or tetrahydrofolates, or derivatives of all the precedingcompounds in which the N-5 or N-10 positions carry one carbon units atvarious levels of oxidation, or pharmaceutically compatible saltsthereof, or a combination of two or more thereof. Exemplarytetrahydrofolates include 5-formyl-(6S)-tetrahydrofolic acid,5-methyl-(6S)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolicacid, 5,10-methenyl-(6R)-tetrahydrofolic acid,10-formyl-(6R)-tetrahydrofolic acid, 5-formimino-(6S)-tetrahydrofolicacid or (6S)-tetrahydrofolic acid, and pharmaceutically acceptable saltsthereof. Exemplary salts include sodium, potassium, calcium or ammoniumsalts.

Exemplary relative weight ratios of BH4 to folates to arginine may befrom about 1:10:10 to about 10:1:1.

The stable formulations of the invention may be provided, e.g. astablets or pills or capsules in HDPE bottles provided with a dessicantcapsule or pouch; or in foil-on-foil blister packaging, or in blisterpackaging comprising see-through polymer film, if commerciallydesirable.

IV. TREATMENT OF BH4-RESPONSIVE DISEASES Hyperphenylalaninemia,Neuropsychological or Neuropsychiatric Disorders

The methods of the invention may be used for treatment of conditionsassociated with elevated phenylalanine levels or decreased tyrosine ortryptophan levels, which may be caused, for example, by reducedphenylalanine hydroxylase, tyrosine hydroxylase, or tryptophanhydroxylase activity. Conditions associated with elevated phenylalaninelevels specifically include phenylketonuria, both mild and classic, andhyperphenylalaninemia as described herein, and exemplary patientpopulations include the patient subgroups described herein as well asany other patient exhibiting phenylalanine levels above normal.

Conditions associated with decreased tyrosine or tryptophan levelsinclude neurotransmitter deficiency, neurological and psychiatricdisorders such as Parkinson's, dystonia, spinocerebellar degeneration,pain, fatigue, depression, other affective disorders and schizophrenia.NO overproduction by nNOS has been implicated in strokes, migraineheadaches, Alzheimer's disease, and with tolerance to and dependence onmorphine. BH4 may be administered for any of these conditions. Otherexemplary neuropsychiatric disorders for which BH4 may be administeredinclude Parkinson's disease, Alzheimer's disease, schizophrenia,schizophreniform disorder, schizoaffective disorder, brief psychoticdisorder, delusional disorder, shared psychotic disorder, psychoticdisorder due to a general medical condition, substance-induced psychoticdisorder, other psychotic disorders, tardive dyskinesia, Machado-Josephdisease, spinocerebellar degeneration, cerebellar ataxia, dystonia,chronic fatigue syndrome, acute or chronic depression, chronic stresssyndrome, fibromyalgia, migraine, attention deficit hyperactivitydisorder, bipolar disease, and autism.

The stable formulations may also be used for treating patients sufferingfrom BH4 deficiency, e.g., due to a defect in the pathway for itssynthesis, including but not limited to dopa-responsive dystonia (DRD),sepiapterin reductase (SR) deficiency, or dihydropteridine reductase(DHPR) deficiency.

Suitable subjects for treatment with the stable formulations of theinvention include subjects with an elevated plasma Phe concentration inthe absence of the therapeutic, e.g. greater than 1800 μM/L, or greaterthan 1600 μM, greater than 1400 μM, greater than 1200 μM, greater than1000 μM, greater than 800 μM, or greater than 600 μM, greater than 420μM, greater than 300 μM, greater than 200 μM, or greater than 180 μM.Mild PKU is generally classified as plasma Phe concentrations of up to600 μM/L, moderate PKU as plasma Phe concentrations of between 600 μM/Lto about 1200 μM/L and classic or severe PKU as plasma Pheconcentrations that are greater than 1200 μM/L. Preferably treatmentwith the stable formulations alone or with protein-restricted dietdecreases the plasma phenylalanine concentration of the subject to lessthan 600 μM, or less than 500 μM, or 360 μM±15 μM or less, or less than200 μM, or less than 100 μM. Other suitable subjects include subjectsdiagnosed as having a reduced phenylalanine hydroxylase (PAH) activity,atypical or malignant phenylketonuria associated with BH4 deficiency,hyperphenylalaninemia associated with liver disorder, andhyperphenylalaninemia associated with malaria. Reduced PAH activity mayresult from a mutation in the PAH enzyme, for example, a mutation in thecatalytic domain of PAH or one or more mutations selected from the groupconsisting of F39L, L48S, I65T, R68S, A104D, S110C, D129G, E178G, V190A,P211T, R241c, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G,A395P, P407S, and Y414C; or subjects that are pregnant females, femalesof child-bearing age that are contemplating pregnancy, or infantsbetween 0 and 3 years of age, or 0-2, 0-1.5 or 0-1; or subjectsdiagnosed as unresponsive within 24 hours to a single-dose BH4 loadingtest or a multiple dose loading test, such as a 4-dose or 7-day loadingtest. Exemplary patient populations and exemplary BH4 loading tests aredescribed in Int'l. Publication No. WO 2005/049000, incorporated hereinby reference in its entirety.

U.S. Pat. Nos. 4,752,573; 4,758,571; 4,774,244; 4,920,122; 5,753,656;5,922,713; 5,874,433; 5,945,452; 6,274,581; 6,410,535; 6,441,038;6,544,994; and U.S. Patent Publications US 20020187958; US 20020106645;US 2002/0076782; US 20030032616 (each incorporated herein by reference)each describe methods of administering BH4 compositions for non-PKUtreatments. Each of those patents is incorporated herein by reference asproviding a general teaching of methods of administering BH4compositions known to those of skill in the art, that may be adapted forthe treatment as described herein.

While individual needs vary, determination of optimal ranges ofeffective amounts of each component is within the skill of the art.Typical dosages of the BH4 comprise about 1 to about 20 mg/kg bodyweight per day, which will usually amount to about 5 (1 mg/kg×5 kg bodyweight) to 3000 mg/day (30 mg/kg×100 kg body weight). While continuous,daily administration is contemplated, for HPA it may be desirable tocease the BH4 therapy when the symptoms of Phe levels are reduced tobelow a certain threshold level. Of course, the therapy may bereinitiated in the event that Phe levels rise again. Appropriate dosagesmay be ascertained through the use of established assays for determiningblood levels of Phe in conjunction with relevant dose response data.

In exemplary embodiments, it is contemplated that the methods of thepresent invention will provide to a patient in need thereof, a dailydose of between about 10 mg/kg to about 20 mg/kg of BH4. Of course, oneskilled in the art may adjust this dose up or down depending on theefficacy being achieved by the administration. The daily dose may beadministered in a single dose or alternatively may be administered inmultiple doses at conveniently spaced intervals. In exemplaryembodiments, the daily dose may be 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg,13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20mg/kg, 22 mg/kg, 24 mg/kg, 26 mg/kg, 28 mg/kg, 30 mg/kg, 32 mg/kg, 34mg/kg, 36 mg/kg, 38 mg/kg, 40 mg/kg, 42 mg/kg, 44 mg/kg, 46 mg/kg, 48mg/kg, 50 mg/kg, or more mg/kg.

Low Dose Regimens

In a low dose therapeutic method of the invention, low doses, e.g.,doses of 0.1 to 5 mg/kg per day are contemplated, including doses of 0.1to 2 mg/kg, or 0.1 to 3 mg/kg, or 1 mg/kg to 5 mg/kg. Doses of less than5 mg/kg per day are preferred. According to the invention, such dosesare expected to provide improvements with relevant study endpoints, andBH4 derivatives are expected to have improved biological propertiesrelative to natural BH4 at such doses. In particular, the inventioncontemplates that any of the1′,2′-diacyl-(6R,S)-5,6,7,8-tetrahydro-L-biopterins or lipoidaltetrahydrobiopterins described herein exhibit improved biologicalproperties at low doses.

The invention also specifically contemplates the use of BH4, or aprecursor or derivative thereof, for treating BH4—responsive diseases ata dose in the range of 0.1 to 5 mg/kg body weight/day, via any route ofadministration including but not limited to oral administration, in aonce daily dose or multiple (e.g. 2, 3 or 4) divided doses per day, fora duration of at least 1, 2, 3, or 4 weeks or longer, or 1, 2, 3, 4, 5,6 months or longer. Exemplary doses include less than 5 mg/kg/day, 4.5mg/kg/day or less, 4 mg/kg/day or less, 3.5 mg/kg/day or less, 3mg/kg/day or less, 2.5 mg/kg/day or less, 2 mg/kg/day or less, 1.5mg/kg/day or less, 1 mg/kg/day or less, or 0.5 mg/kg/day or less.Equivalent doses per body surface area are also contemplated.

For the person of average weight/body surface area (e.g. 70 kg), theinvention also contemplates a total daily dose of less than 400 mg.Exemplary such total daily doses include 360 mg/day, 350 mg/day, 300mg/day, 280 mg/day, 210 mg/day, 180 mg/day, 175 mg/day, 150 mg/day, or140 mg/day. For example, 350 mg/day or 175 mg/day is easilyadministrable with an oral dosage formulation of 175 mg, once or twice aday. Other exemplary total daily doses include 320 mg/day or less, 160mg/day or less, or 80 mg/day or less. Such doses are easilyadministrable with an oral dosage formulation of 80 or 160 mg. Otherexemplary total daily doses include 45, 90, 135, 180, 225, 270, 315 or360 mg/day or less, easily administrable with an oral dosage formulationof 45 or 90 mg. Yet other exemplary total daily doses include 60, 120,180, 240, 300, or 360 mg/day, easily administrable with an oral dosageformulation of 60 or 120 mg. Other exemplary total daily doses include70, 140, 210, 280, or 350 mg/day, easily administrable with an oraldosage formulation of 70 or 140 mg. Exemplary total daily doses alsoinclude 55, 110, 165, 220, 275 or 330 mg/day, easily administrable withan oral dosage formulation of 55 mg. Other exemplary total daily dosesinclude 65, 130, 195, 260, or 325 mg/day, or 75, 150, 225, 300 or 375mg/day, e.g. in dosage formulations of 65 mg or 75 mg.

Diseases Associated with Nitric Oxide Synthase Dysfunction

The invention further contemplates that stable formulations of theinvention may be used for treatment of subjects suffering fromconditions that would benefit from enhancement of nitric oxide synthaseactivity and patients suffering from vascular diseases, ischemic orinflammatory diseases, or insulin resistance. The treatment may, forexample alleviate a deficiency in nitric oxide synthase activity or may,for example provide an increase in nitric oxide synthase activity overnormal levels. It has been suggested that a patient suffering from adeficiency in nitric oxide synthase activity would benefit fromco-treatment with folates, including folate precursors, folic acids, orfolate derivatives.

Nitric oxide is constitutively produced by vascular endothelial cellswhere it plays a key physiological role in the regulation of bloodpressure and vascular tone. It has been suggested that a deficiency innitric oxide bioactivity is involved in the pathogenesis of vasculardysfunctions, including coronary artery disease, atherosclerosis of anyarteries, including coronary, carotid, cerebral, or peripheral vasculararteries, ischemia-reperfusion injury, hypertension, diabetes, diabeticvasculopathy, cardiovascular disease, peripheral vascular disease, orneurodegenerative conditions stemming from ischemia and/or inflammation,such as stroke, and that such pathogenesis includes damaged endothelium,insufficient oxygen flow to organs and tissues, elevated systemicvascular resistance (high blood pressure), vascular smooth muscleproliferation, progression of vascular stenosis (narrowing) andinflammation. Thus, treatment of any of these conditions is contemplatedaccording to methods of the invention.

It has also been suggested that the enhancement of nitric oxide synthaseactivity also results in reduction of elevated superoxide levels,increased insulin sensitivity, and reduction in vascular dysfunctionassociated with insulin resistance, as described in U.S. Pat. No.6,410,535, incorporated herein by reference. Thus, treatment of diabetes(type I or type II), hyperinsulinemia, or insulin resistance iscontemplated according to the invention. Diseases or disorders havingvascular dysfunction associated with insulin resistance include thosecaused by insulin resistance or aggravated by insulin resistance, orthose for which cure is retarded by insulin resistance, include but arenot limited to abnormal vascular compliance, endothelial dysfunction andhypertension, disorders of insulin sensitivity and glucose control,abnormal peripheral perfusion such as intermittent claudication, reducedperipheral perfusion, decreased skin blood flow, defective wound healingand peripheral circulation disorder, hyperlipidemia, arteriosclerosis,coronary vasoconstrictive angina, effort angina, cerebrovascularconstrictive lesion, cerebrovascular insufficiency, cerebral vasospasm,coronary arteriorestenosis following percutaneous transluminal coronaryangioplasty (PTCA) or coronary artery bypass grafting (CABG), obesity,insulin-independent diabetes, hyperinsulinemia, lipid metabolismabnormality, coronary arteriosclerotic heart diseases, congestive heartfailure, pulmonary hypertension with or without congestive heartfailure, exercise-associated angina, coronary artery disease and relatedatherosclerosis; ophthalmic disease such as optic atrophy and diabeticretinal disease; and renal disease such as microalbuminuria in diabeticrenal disease, renal failure and decreased glomerular filtration rate.

It is contemplated that when administered to patients with thesediseases, BH4 can prevent or treat these diseases by activating thefunctions of NOS, increasing NO production and suppressing theproduction of active oxygen species to improve disorders of vascularendothelial cells.

The invention provides a method for treating a subject diagnosed ashaving vascular disease unrelated to diabetes selected from the groupconsisting of pulmonary vascular disease, hemolytic anemias, stroke andrelated ischemic vascular disease (such as stroke, cardiac or coronarydisease, arteriosclerosis, or peripheral vascular disease), thrombosis,transplant-related endothelial dysfunction, and cardiac or coronarydisease. In one embodiment, pulmonary vascular disease includes but isnot limited to pulmonary tension in sickle cell anemia and otherhemoglobinopathies, idiopathic pulmonary hypertension, persistentpulmonary hypertension of the newborn (PPHN). In a further embodiment,hemolytic anemias include hereditary hemolytic anemias and acquiredhemolytic anemia. Hereditary hemolytic anemias include but are notlimited to sickle-cell anemia, thalassemia, hemolytic anemia due to G6PDdeficiency, pyruvate kinase deficiency, hereditary elliptocytosis,hereditary spherocytosis, hereditary stomatocytosis, hereditaryovalocytosis, paroxysmal nocturnal hemoglobinuria, and hemoglobin SCdisease. Acquired hemolytic anemias include but are not limited tomicroangiopathic hemolytic anemia, idiopathic autoimmune hemolyticanemia, non-immune hemolytic anemia caused by chemical or physicalagents or devices (left ventricular assist devices), mechanical heartvalves and bypass devices), and secondary immune hemolytic anemia.

In another embodiment, stroke and related ischemic vascular diseaseincludes but is not limited to vasospasm, such as post-strokecerebrovascular spasm. Thrombosis includes but is not limited tothrombogenesis, thrombosis, clotting, and coagulation. In a furtherembodiment, transplant-related endothelial dysfunction includes but isnot limited to vascular dysfunction after solid organ transplantationand cyclosporine A induced endothelial dysfunction. In yet anotherembodiment, cardiac or coronary disease includes but is not limited tocongestive heart failure, vascular dysfunction and angina associatedwith hypercholesterolemia, and vascular dysfunction and anginaassociated with tobacco smoking.

BH4 can also prevent or treat other disorders associated with theoverproduction of or damage related to reactive oxygen species,including but not limited to sepsis.

It is understood that the suitable dose of a composition according tothe present invention will depend upon the age, health and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired (i.e., the amount of decrease inplasma Phe concentration desired). The frequency of dosing also isdependent on pharmacodynamic effects on Phe levels. If the effect lastsfor 24 hours from a single dose. However, the most preferred dosage canbe tailored to the individual subject, as is understood and determinableby one of skill in the art, without undue experimentation. Thistypically involves adjustment of a standard dose, e.g., reduction of thedose if the patient has a low body weight.

The frequency of BH4 dosing will depend on the pharmacokineticparameters of the agent and the routes of administration. The optimalpharmaceutical formulation will be determined by one of skill in the artdepending on the route of administration and the desired dosage. See forexample Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publ.Co, Easton Pa. 18042) pp 1435 1712, incorporated herein by reference.Such formulations may influence the physical state, stability, rate ofin vivo release and rate of in vivo clearance of the administeredagents. Depending on the route of administration, a suitable dose may becalculated according to body weight, body surface areas or organ size.Further refinement of the calculations necessary to determine theappropriate treatment dose is routinely made by those of ordinary skillin the art without undue experimentation, especially in light of thedosage information and assays disclosed herein as well as thepharmacokinetic data observed in animals or human clinical trials.

The final dosage regimen will be determined by the attending physician,considering factors which modify the action of drugs, e.g., the drug'sspecific activity, severity of the damage and the responsiveness of thepatient, the age, condition, body weight, sex and diet of the patient,the severity of any infection, time of administration and other clinicalfactors. As studies are conducted, further information will emergeregarding appropriate dosage levels and duration of treatment forspecific diseases and conditions.

V. COMBINATION THERAPY

Certain methods of the invention involve the combined use of the stableformulations of the invention and one or more other therapeutic agents.

In such combination therapy, administration of the stable formulationsof the invention may be concurrent with or may precede or follow theadministration of the second therapeutic agent, e.g. by intervalsranging from minutes to hours, so long as both agents are able to exerttheir therapeutic effect at overlapping time periods. Thus, theinvention contemplates the stable formulations of the invention for usewith a second therapeutic agent. The invention also contemplates use ofa second therapeutic agent in preparation of a medicament foradministration with the stable tetrahydrobiopterin, precursor,derivative or analog formulations of the invention.

Tetrahydrobiopterin therapy may be combined with dietary proteinrestriction to effect a therapeutic outcome in patients with variousforms of HPA. For example, one could administer to the subject the BH4composition and a low-phenylalanine medical protein composition in acombined amount effective to produce the desired therapeutic outcome(i.e., a lowering of plasma Phe concentration and/or the ability totolerate greater amounts of Phe/protein intake without producing aconcomitant increase in plasma Phe concentrations). This process mayinvolve administering the BH4 composition and the dietary proteintherapeutic composition at the same time. This may be achieved byadministering a single composition or pharmacological proteinformulation that includes all of the dietary protein requirements andalso includes the BH4 within said protein formulation. Alternatively,the dietary protein (supplement or normal protein meal) is taken atabout the same time as a pharmacological formulation (tablet, injectionor drink) of BH4.

In some embodiments, the protein-restricted diet is one which issupplemented with amino acids, such as tyrosine, valine, isoleucine andleucine. The patient may be co-administered a low-Phe proteinsupplement, which may include L-tyrosine, L-glutamine, L-carnitine at aconcentration of 20 mg/100 g supplement, L-taurine at a concentration of40 mg/100 g supplement and selenium. It may further comprise therecommended daily doses of minerals, e.g., calcium, phosphorus andmagnesium. The supplement further may comprise the recommended dailydose of one or more amino acids selected from the group consisting ofL-leucine, L-proline, L-lysine acetate, L-valine, L-isoleucine,L-arginine, L-alanine, glycine, L-asparagine monohydrate, L-tryptophan,L-serine, L-threonine, L-histidine, L-methionine, L-glutamic acid, andL-aspartic acid. In addition, the supplement may be fortified with therecommended daily dosage of vitamins A, D and E. Optionally, thesupplement comprises a fat content that provides at least 40% of theenergy of the supplement. Such supplements may be provided in the formof a powder supplement or in the form of a protein bar. In certainembodiments, protein-restricted diet comprises a protein supplement andthe BH4 is provided in the same composition as the protein supplement.

In other alternatives, the BH4 treatment may precede or follow thedietary protein therapy by intervals ranging from minutes to hours. Inembodiments where the protein and the BH4 compositions are administeredseparately, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the BH4 willstill be able to exert an advantageously effect on the patient. In suchinstances, it is contemplated that one would administer the BH4 withinabout 2-6 hours (before or after) of the dietary protein intake, forexample, with a delay time of only about 1 hour or less. In certainembodiments, it is contemplated that the BH4 therapy will be acontinuous therapy where a daily dose of BH4 is administered to thepatient indefinitely. In other situations, e.g., in pregnant womenhaving only the milder forms of PKU and HPA, it may be that the BH4therapy is only continued for as long as the woman is pregnant and/orbreast feeding.

Further, in addition to therapies based solely on the delivery of BH4and dietary protein regulation, the methods of the present inventionalso contemplate combination therapy with a third composition thatspecifically targets one or more of the symptoms of HPA. For example, itis known that the deficit in tyrosine caused by HPA results in adeficiency in neurotransmitters dopamine and serotonin. Thus, in thecontext of the present invention, it is contemplated that BH4 anddietary protein based methods could be further combined withadministration of L-dopa, carbidopa and 5-hydroxytryptophanneurotransmitters to correct the defects that result from decreasedamounts of tyrosine in the diet.

In addition, gene therapy with both PAH (Christensen et al., Mol. Gent.And Metabol. 76: 313-318, 2002; Christensen et al., Gene Therapy,7:1971-1978, 2000) and phenylalanine ammonia-lyase (PAL Liu et al.,Arts. Cells. Blood. Subs and Immob. Biotech. 30(4)243-257, 2002) hasbeen contemplated by those of skill in the art. Such gene therapytechniques could be used in combination with the combined BH4/dietaryprotein restriction based therapies of the invention. In furthercombination therapies, it is contemplated that phenylase may be providedas an injectable enzyme to destroy lower Phe concentrations in thepatient. As the administration of phenylase would not generate tyrosine(unlike administration of PAH), such treatment will still result intyrosine being an essential amino acid for such patients. Thereforedietary supplementation with tyrosine may be desirable for patientsreceiving phenylase in combination with the BH4 therapy.

BH4 may be co-administered for neuropsychological or neuropsychiatricdisorders according to the method of the invention with one or moreother neuropsychiatric active agents, including antidepressants,neurotransmitter precursors such as tryptophan, tyrosine, serotonin,agents which activate noradrenergic systems, such as lofepramine,desipramine, reboxetine, tyrosine, agents which act preferentially onserotonin, combined inhibitors of both noradrenaline and serotoninuptake, such as venlafaxine, duloxetine or milnacipran, or drugs whichare combined inhibitors of both dopamine and noradrenaline reuptake suchas bupropion.

In a related embodiment, BH4 is administered with other therapeuticagents commonly used to treat diabetes, vascular disease,hyperlipidemia. Agents used to treat diabetes, include but not limitedto agents that improve insulin sensitivity such as PPAR gamma ligands(thiazolidinedones, glitazones, troglitazones, rosiglitazone (Avandia),pioglitazone), stimulators of insulin secretion such as sulphonylureas(gliquidone, tolbutamide, glimepride, chlorpropamide, glipizide,glyburide, acetohexamide) and meglitinides (meglitinide, repaglinide,nateglinide) and agents that reduce liver production of glucose such asmetformin. Agent used to treat vascular disease, include but not limitedto endothelin receptor antagonists commonly used for the treatment ofhypertension and other endothelial dysfunction-related disorders, suchas bosentan, darusentan, enrasentan, tezosentan, atrasentan, ambrisentansitaxsentan; smooth muscle relaxants such as PDE5 inhibitors(indirect-acting) and minoxidil (direct-acting); angiotensin convertingenzyme (ACE) inhibitors such as captopril, enalapril, lisinopril,fosinopril, perindopril, quinapril, trandolapril, benazepril, ramipril;angiotensin II receptor blockers such as irbesartan, losartan,valsartan, eprosartan, olmesartan, candesartan, telmisartan; betablockers such as atenolol, metoprolol, nadolol, bisoprolol, pindolol,acebutolol, betaxolol, propranolol; diuretics such ashydrochlorothiazide, furosemide, torsemide, metolazone; calcium channelblockers such as amlodipine, felodipine, nisoldipine, nifedipine,verapamil, diltiazem; alpha receptor blockers doxazosin, terazosin,alfuzosin, tamsulosin; and central alpha agonists such as clonidine.Agents used to treat hyperlipidemia, include but not limited to agentsthat lower LDL such as statins (atorvastatin, fluvastatin, lovastatin,pravastatin, rosuvastatin calcium, simvastatin) and nicotinic acid,cholesteryl ester transfer protein inhibitors (such as torcetrapib),agents that stimulate PPAR alpha such as fibrates, gemfibrozil,fenofibrate, bezafibrate, ciprofibrate, agents that bind and preventreadsorption of bile acids and reduce cholesterol levels such as bileacid sequestrants, cholestyramine and colestipol, and cholesterolabsorption inhibitors.

BH4 may also be administered with a factor or combination of factorsthat enhances or normalizes the production of the vasodilator nitricoxide (NO) alone or in combination with a therapeutic agent. In oneembodiment, such factor(s) enhances the activity or expression the denovo biosynthesis of BH4 and is selected from the group consisting ofguanosine triphosphate cyclohydrolase I (GTPCH1),6-pyruvoyltetrahydropterin synthase (PTPS) and sepiapterin reductase. Ina preferred embodiment of the invention, BH4 synthesis is increased byincreasing the expression of GTPCH1 expression by the use of any one ormore cyclic adenosine monophosphate (cAMP) analogs or agonists includingforskolin, 8-bromo cAMP or other agents that function to increase cAMPmediated cell signaling, for example, cytokines and growth factorsincluding interleukin-1, interferon-gamma (IFN-γ), tumor necrosis factoralpha (TNF-α), c-reactive protein, HMG-CoA-reductases (statins likeatorvastatin) nerve growth factor (NGF), epidermal growth factor (EGF),hormones including adrenomedullin and estradiol benzoate, and othercompounds such as NADPH and NADPH analogs, caffeine, cyclosporine Amethyl-xanthines including 3-isobutyl-1-methyl xanthine, theophylline,reserpine, hydrogen peroxide.

One embodiment of invention therefore relates to increasing GTPCH1levels by inhibiting the degradation of 3′5′-cyclic nucleotides usinginhibitors of the eleven phosphodiesterases families (PDE1-11) includingPDE1, PDE3, PDE5. The PDE inhibitors of the present invention includeViagra/sildanefil, cialis/tadalafil, vardenafil/levitra, udenafil,8-Methoxymethyl-IBMX, UK-90234, dexamethasone, hesperetin, hesperedins,Irsogladine, vinpocetine, cilostamide, rolipram, ethylbeta-carboline-3-carboxylate (beta-CCE), tetrahydro-beta-carbolinederivatives, 3-O-methylquercetin and the like.

Another embodiment of the invention relates to increasing the levels ofBH4 by increasing the levels of BH4-synthesizing enzymes by gene therapyor endothelium-targeted delivery of polynucleotides of the syntheticmachinery of BH4. Yet another embodiment of the invention relates toincreasing the levels of BH4 by supplementation with BH4-synthesizingenzymes GTPCH1, PTPS, SR, PCD, DHPR and DHFR. It is contemplated thatBH4-synthesizing enzymes encompasses all natural and unnatural forms ofthe enzymes including mutants of the proteins.

Another embodiment of the invention relates to increasing BH4 levels bydiverting the substrate 7,8-dihydroneopterin triphosphate towards BH4synthesizing enzyme PTPS instead of alkaline phosphatase (AP) byinhibiting AP activity. The agents or compounds that inhibit theactivity of AP include phosphate analogs, levamisole, and L-Phe. Anotherembodiment of the invention relates to agents or compounds that inhibitalkaline phosphatase includes the small inhibitory RNA (siRNA),antisense RNA, dsDNA, small molecules, neutralizing antibodies, singlechain, chimeric, humanized and antibody fragments to inhibit thesynthesis of alkaline phosphatase.

Another embodiment of the invention includes agents or compounds thatenhance the activity of catalysts or cofactors needed for the synthesisof enzymes of the de novo synthesis pathway of BH4 synthesis.

Another embodiment of the invention includes agents or compounds thatprevent the degradation of the enzymes needed for the synthesis of BH4.Yet another embodiment of the invention includes agents or compoundsthat prevent the degradation of the catalysts needed for the synthesisof BH4 and its synthetic enzymes including GTPCH1, PTPS and SR.

Another embodiment of the invention relates to increasing the levels ofBH4 by increasing the reduction of BH2 via the salvage pathway. In vivo,BH4 becomes oxidized to BH2. BH2 which exist as the quinoid form (qBH2)and as the 7,8-dihydropterin which is reduced to BH4 by DHPR and DHFRrespectively. One embodiment of the invention relates to increasing theregeneration or salvage of BH4 from BH2 by modulating the activity andsynthesis of the enzymes PCD, DHPR and DHFR using agents or compoundsthat pathway NADPH, thiols, perchloromercuribenzoate, hydrogen peroxideand the like.

Another embodiment of the invention relates to agents that stabilize BH4by decreasing the oxidation of BH4 using agents or compounds such asantioxidants including ascorbic acid (vitamin C), alpha tocopherol(vitamin E), tocopherols (e.g vitamin A), selenium, beta-carotenes,carotenoids, flavones, flavonoids, folates, flavones, flavanones,isoflavones, catechins, anthocyanidins, and chalcones.

In a further embodiment, such factor(s) may increase the activity orexpression of nitric oxide synthase and thereby enhance the generationof NO.

In yet another embodiment, the invention contemplates factors thatinhibit the GTPCH feedback regulatory protein, GFRP. An embodiment ofthe invention relates to agents or compounds that inhibit the binding ofBH4 to the GTPCH1/GFRP complex, thereby preventing the feedbackinhibition by BH4. Agents or compounds of this invention includecompetitive inhibitors such as alternate forms of BH4 with alteredaffinities for the complex, structural analogs etc. Still anotherembodiment of the invention includes agents or compounds that enhancethe binding of L-phenylalanine to CTPCH1/GFRP inducing the synthesis ofBH4. Another embodiment of the invention includes agents or compoundsthat increase the levels of L-Phe such as precursors of L-Phenylalanie,which serves to inhibit the feedback inhibition of GTPCH1 by GFRP andBH4.

Yet another embodiment of the invention relates to agents or compoundsthat modulate the activity or the synthesis of GFRP. An embodiment ofthe invention includes agents or compounds that inhibit the activity ofGFRP. Another embodiment of the invention includes the use of siRNA,small molecules, antibodies, antibody fragments and the like to inhibitthe synthesis of GFRP.

VI. BIOPTERIN ASSAYS

The concentration of total biopterin and oxidized biopterin in plasma,blood and other tissues are determined based on the method of Fukishimaet al (Anal. Biochem. 102:176 (1980). Biopterin has four different formsincluding two forms of reduced biopterin, R-tetrahydrobiopterin (BH4)and quinonoid R-dihydrobiopterin (q-BH2) and two forms of oxidizedbiopterin, dihydrobiopterin (BH2) and biopterin (B). Of these fourforms, only the reduced forms of biopterin have coenzymatic activity.Reduced biopterin is converted to B by iodylation under acidicconditions, whereas under alkaline conditions, it is converted topterin. Oxidized biopterin is converted to B by iodylation under acidicand alkaline conditions. By taking advantage of this property, theamount of total biopterin is determined upon iodylation under acidicconditions and that of oxidized biopterin is determined upon iodylationunder alkaline conditions, so that the amount of reduced biopterin iscalculated from the difference in quantity thereof. When used as acoenzyme, BH4 is converted to q-BH2. The q-BH2 is immediately convertedto BH4 by dihydropterine reductase or if not reduced, it is oxidized toBH2 or DHPT. Because it is difficult for biopterin to exist in the formof q-BH2 in vivo, the reduced biopterin may well be displaced as BH4.

Plasma and whole blood samples collected are immediately subjected tooxidation with acidic oxidizing solution (0.6N HCl solution in watercontaining 0.6% potassium iodide (KI), 0.3% iodine (I2) and 0.6Ntrichloroacetic acid (TCA)) and alkaline oxidizing solution (0.7N sodiumhydroxide (NaOH)). Determination of B is performed by HPLC andradioactivity is measured using a liquid scintillation counter.

Measurement of BH4 using Reverse Phase HPLC(RP) Coupled with Tandem MassSpectrometry (LC/MS/MS): The combined use of reverse phase highperformance liquid chromatography (RP) and tandem mass spectrometry(LC/MS/MS) was shown to be selective for BH4 in human plasma, sensitivefor BH4 in the range of 5-1000 ng/mL. The method is associated withabout 50% conversion of BH4 due to oxidation during collection andstorage. Samples are stable for greater than 3 months in dipotassiumsalt of ethylenediaminetetraacetic acid (K₂EDTA) plasma. Recovery fromthe pretreatment steps is about 75%. The accuracy and precision of themethod was determined to have coefficient of variation (CV) % below 15%(20% at the lower limit of quantitation, LLOQ).

The combined use of HPLC and tandem mass spectrometry was shown to be animprovement over HPLC alone in determining the BH4 test article becauseof: (1) its increased selectivity for drug-BH4 (whereas HPLC measurestotal biopterin), (2) broader qualitative range, (3) establishedconversion ration, (4) extensive characterization and proven utility inhuman subjects, and (5) novel and useful measurement in differentspecies and matrices.

The improved method comprises the following steps. Samples of blood,plasma, tissue homogenates, or urine are subjected to acidic or alkalineoxidation. With acidic oxidation, (1) the samples are treated withpotassium chloride (KCl), hydrochloric acid (HCl) or TCA for an hour;(2) the acid oxidized samples are then subjected to iodometry; (3) thesamples are run through an ion exchange column; (4) total biopterincomprising BH4, q-BH2 (which is immediately reduced in vivo to BH4 suchthat the measured reduced biopterin is based mainly upon BH4), BH2, andB are measured using HPLC and tandem mass spectrometry. With alkalineoxidation, (1) the samples are treated with KI, I2 or NaOH for an hour;(2) the alkaline oxidized samples are then subjected to acidificationwith HCl or TCA; (3) subjected to iodometry; (4) the samples are runthrough an ion exchange column; (5) oxidized biopterin comprising BH2and B are measured; (6) different species are measured using HPLC andtandem mass spectrometry; and (7) the amount of reduced biopterin(BH4+q-BH2) is calculated as the difference between total biopterinsless the oxidized form.

Flow charts for biopterin measurement and assay validation summary areprovided in FIGS. 16 and 17.

Optimized Assay

An HPLC method using Electrochemical Detection (ECD) and Fluorescence(FL) detection is advantageous as it allows for the measurement of eachof the discrete biopterin compounds (BH4, BH2 and B) as well as analogs.

BH4 is a cofactor for the enzyme system nitric oxide synthase (NOS),which produces nitric oxide (NO). The production of NO is important formaintaining vascular homeostasis. When intracellular levels of BH4 arelimited, NO production is diminished (due to decreased NOS activity) andleads to the generation of the damaging free radical superoxide (O₂—).Excess O₂— can lead to endothelial dysfunction and may contribute to theoxidation of BH4 to BH2. A low ratio of BH4 to BH2 may promoteendothelial injury, whereas a high BH4 to BH2 ratio may promoteendothelial health. Therefore, characterizing the BH4 to BH2 ratio mayserve as a predictor of endothelial health.

The concentrations of different biopterins (BH4, BH2 and B) or analogsare determined by initially using reverse phase HPLC for separation,followed by ECD and FL detection.

BH4, which is a redox-sensitive, non-fluorescent molecule, is measuredusing ECD. BH4 (and analogs thereof) are measured using ECD in which BH4(or analog) is oxidized by electrode 1 to a quinonoid dihydrobiopterinform (e.g., qBH2), a short-lived dihydrobiopterin intermediate, which isthen reduced back to BH4 (or analog) at electrode 2. The detector thenuses the current generated by this reduction reaction to determine theconcentration of BH4 or analog thereof (endogenous qBH2 is negligible).

BH2, B, and analogs thereof can be measured in the same injection byfluorescence detection. Post-ECD oxidation of BH2 or an analog thereofusing a conditioning guard cell at the optimum potential oxidizes BH2 oran analog thereof to B or the corresponding biopterin analog. This isdesirable because BH2 is not fluorescently active or easily measured andmust be converted to B, which is easily measured using fluorescence.Endogenous BH2, once converted to B, and endogenous B are distinguishedfrom one another by two separate fluorescent peaks, due to the differentretention times on the HPLC column for each molecule.

In total the methods can be used to measure the species BH4, BH2, and B,and analogs thereof. The biopterins preferably are measured using a 2%MeOH-containing mobile phase, as described herein. Biopterin analogs,such as valine biopterin derivatives, may be better suited to highermethanol contents in the mobile phase, e.g. a 10% MeOH-containing mobilephase.

Thus, a method for detecting biopterins in a mixture of biopterinspecies can include (a) separating biopterin species in the mixture byreverse phase HPLC; and in the case of BH4 and analogs thereof, (b1)performing electrochemical detection by oxidizing the BH4 and analogsthereof present by a first electrode to quinonoid dihydrobiopterinforms, followed by reducing the quinonoid forms back to BH4 and analogsthereof present at a second electrode, and measuring current generatedby the reduction reaction to determine the concentration of species;and/or (b2) in the case of BH2, analogs thereof, biopterin, or analogsthereof, measuring such species by fluorescence detection followingpost-column oxidation of BH2 species to biopterin. Preferably, themobile phase is one disclosed herein.

In one embodiment, the preferred mobile phase includes sodium acetate,citric acid, EDTA, and 1,4-dithioerythritol (DTE) with methanol.Preferred concentrations are 50 mM sodium acetate, 5 mM citric acid, 48μM EDTA, and 160 μM DTE with 2% methanol.

VII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Concentration Time Curve for Biopterin in Plasma after aSingle Oral Dose in the Rat

The purpose of this study was to assess the pharmacokinetics of BH4after a single oral administration in rats. Single doses of BH4 (10 and100 mg/kg) were administered orally to male Sprague Dawley rats (6 weeksold) under fasting conditions.

Results

The maximum total biopterin concentrations in plasma 2 hrs and 1 hrpost-dosing were 108 ng/ml (i.e., about 3× the endogenous level) and1227 ng/ml (i.e., about 30× the endogenous level), respectively (FIG.18). Thereafter, biopterin had an elimination half-life (t_(1/2)) ofabout 1.1 hr, returning to the endogenous level 9 hrs post-dosing forthe 10 mg/kg dose and 24 hrs post-dosing for the 100 mg/kg dose (FIG.18).

The bioavailability (F) after a 10 and 100 mg/kg oral administrationwere 6.8% and 11.8%, respectively, based on the area under the plasmaconcentration-time curve (ΔAUC) obtained by subtracting the endogenouslevel during a 10 mg/kg intravenous administration. Rate of GIabsorption were 8.8% when measured using radioactive markers in urine.An estimate of the actual value would be approximately 10% oralbioavailability based on these data.

The ratio of reduced biopterin to total biopterins in plasma (i.e., thereduced-form ratio) was relatively static (73%-96%) (FIG. 19).

Example 2 Concentration Time Curve for Biopterin in Plasma after SingleOral Dose to Monkey

The purpose of this study was to assess pharmacokinetics of sapropterinafter a single oral administration in cynomolgus monkeys. A single doseof sapropterin (10 mg/kg) was administered orally to female cynomolgusmonkeys (3/group) under fasting conditions.

Results

The total plasma biopterin concentration (ΔC) reached its maximum value3 hrs post-dosing (344 ng/ml, approximately 20× endogenous levels) (FIG.20). The plasma elimination half-life of biopterin was approximately 1.4hrs, returning to the endogenous level within 24 hrs post-dosing. Theratio of reduced biopterin to total biopterins was nearly constantduring the test period. The bioavailability (F) following a 10 mg/kgoral administration to female monkeys was about 9%, measured as ΔAUCoral/iv ratios (FIG. 21).

Example 3 Relative Bioavailability of Tetrahydrobiopterin (BH4)Administered after Dissolution of Tablet(s) in Water or Administered asIntact Tablet(s), and Effect of Food on Absorption in Healthy SubjectsObjectives

The primary objectives of the study were: (1) to evaluate the relativebioavailability of tetrahydrobiopterin (BH4, sapropterindihydrochloride) when administered after dissolution of tablet(s) inwater or administered as intact tablet(s); (2) to compare the effect offood on the bioavailability of BH4 in healthy subjects. The secondaryobjective of the study was to assess the safety and tolerability ofsingle oral doses of BH4 in healthy subjects.

Methodology

This study was an open-label, randomized, three-treatment, six-sequence,three-period crossover study in which 30 subjects were to complete 3single-dose dosing periods and were randomized to one of six sequencegroups (Groups 1, 2, 3, 4, 5, and 6):

Group 1: a, b, c

Group 2: b, c, a

Group 3: c, a, b

Group 4: a, c, b

Group 5: b, a, c

Group 6: c, b, a

where all dosing groups received BH4 10 mg/kg orally as follows:a: administered after dissolution of tablet(s) in water given in fastingunder fasting conditionsb: administered as intact tablet(s) given in fasting under fastingconditionsc: administered as intact tablet(s) given 30 minutes after beginning toingest a high-calorie, high-fat meal in fed conditions

Each subject received a single dose of 10 mg/kg of BH4 during eachtreatment period. A washout period of at least seven days separated eachdose administration. A post-study assessment was performed 5-7 daysafter discharge of the third treatment period. Blood samples forPharmacokinetic (PK) analysis were drawn at scheduled collection timesduring each study period: within 30 minutes prior to dose, and 0.5, 1.0,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 8.0, 10.0, 12.0, 18.0, and 24.0hours post-dose.

Dose and Mode of Administration

BH4 tablets were administered as 10 mg/kg dosages per treatment period.Tablets were administered by a) dissolution in water given in a fastedstate, b) as intact tablets given in a fasted state, or c) as intacttablets given in a fed state.

Each dose of study drug was prepared and administered in liquid(solution) form mixed with water. The water supplied was ambienttemperature tap water. Dosing solutions were prepared within 15 minutesof scheduled dose time. Dissolution of the tablet in liquid tookapproximately 1 to 3 minutes. The tablets were broken up or crushed inthe dosing cup prior to dissolution in order to improve dissolutionrate.

At the designated morning dosing time, BH4 was administered orally asthe number of tablets equivalent to a 10 mg/kg dose, dissolved in 120 mLof water or orange juice. Each subject was observed closely as theentire 120 mL dose was consumed within 15 minutes of preparation.Immediately after the dose had been consumed, the dosing cup was rinsedwith 60 mL of water and the subject consumed the rinse. A second 60 mLwater rinse was added to the dosing cup and then the subject consumedthe second rinse. The entire dosing procedure was completed in a1-minute time period. A qualified staff person inspected the dosing cupand each subject's mouth immediately after completion of the dose toensure that the entire dose was consumed. Alternatively, the subjectswallowed a pill containing the BH4 rather than dissolving it in water.For each individual, the dosing periods occurred with a minimum of 7days between doses.

Food Intake Schedule

A snack was served the evening of check-in. All subjects were thenrequired to fast for at least 10 hours prior to dosing.

Fasting Conditions

Subjects receiving treatments administered under fasting conditions weredosed after they completed a minimum 10-hour overnight fast.

The subjects continued to fast for 4 hours post dose. Water was allowedad lib during the study except for 1 hour prior through 1 hourpost-dose. Standardized meals were provided at approximately 4 and 10hours after drug administration and at appropriate times thereafter.

Non-Fasting Conditions

Subjects receiving treatments administered under non-fasting conditionswere dosed after consuming a high-calorie, high-fat breakfast meal.Subjects received the following standard high-fat (approximately 50% oftotal caloric content of the meal), high-calorie (approximately 1000calories) breakfast that began 30 minutes prior to scheduledadministration of the dose and ended (last bite taken) within 5 minutesprior to dosing.

2 eggs fried in butter

2 strips of bacon

2 slices of toast with butter

4 ounces of hash brown potatoes

8 ounces of whole milk

This meal contained approximately 150 protein calories, 250 carbohydratecalories, and 500-600 fat calories. An equivalent meal was substitutedwith documentation of the menu and caloric contents.

The subjects then fasted for 4 hours post dose. Water was allowed ad libduring the study except for 1 hour prior through 1 hour post-dose.Standard meals were provided at approximately 4 and 10 hours after drugadministration and at appropriate times thereafter.

Duration of Treatment

Three single-dose treatment periods were each separated by a minimum of7 days.

A follow-up visit was conducted 5 to 7 days after the last treatmentvisit.

Safety Variables: Evaluation and Methods

Safety was evaluated for all subjects who take at least one dose of BH4.

Efficacy and Safety Measurements Assessed and Flow Chart

Safety was evaluated by recording the incidence of adverse events,changes in 12-lead ECG parameters, vital signs and physical examinationresults, and changes in baseline in laboratory test values. The schedulefor these assessments is shown in FIG. 22.

Physical Examinations and Vital Signs

Each subject underwent a routine physical examination by the studyinvestigator. The physical examination included evaluation of head,eyes, ears, nose, throat, neck, heart, chest, lungs, abdomen,extremities, peripheral pulses, neurologic status, skin, and otherphysical conditions of note are evaluated. This study protocol did notrequire genitourinary examinations.

Height (in centimeters) and weight (in kilograms) were measured and bodymass index (BMT) was calculated (BMT=weight (kg)/[height(m)]²).

Blood pressure was measured in the sitting position according to theAmerican Heart Association recommendations. Subjects were at rest withtheir feet on the floor for 5 minutes in the sitting position when bloodpressure was measured.

Heart (pulse) rate was measured while the subject was in the sittingposition.

A standardized 12-lead electrocardiogram (ECG) recording was taken atscreening and at study discharge. ECGs were evaluated by a qualifiedinvestigator. Copies of the ECG and evaluation reports were kept as partof each subject's file.

The medical history, clinical laboratory test results and ECG tracing(s)were reviewed and evaluated by the Principal Investigator to determineclinical eligibility of each subject to participate in the study.

Clinical Laboratory Assessments Hematology:

The following were evaluated: hemoglobin, hematocrit, total anddifferential leukocyte count, red blood cell (RBC) and platelet count.

In addition, blood was tested for Hepatitis B Surface Antigen, HepatitisC Antibody and Human Immunodeficiency Virus (HIV).

Chemistry:

The following were evaluated: albumin, blood urea nitrogen (BUN),creatinine, total bilirubin, alkaline phosphatase (ALP), aspartatetransaminase (AST), alanine transaminase (ALT), sodium (Na⁺), potassium(K⁺), chloride (Cl⁻), lactic dehydrogenase (LDH), uric acid, andglucose.

Urinalysis:

The following were evaluated by the urine dipstick method: pH, specificgravity, protein, glucose, ketones, bilirubin, blood, nitrite, andurobilinogen. If protein, occult blood, or nitrite values are out ofrange, a microscopic examination is performed.

Urine samples were also tested for drugs of abuse (amphetamines,barbiturates, benzodiazepines, cannabinoids, cocaine and opiates).

Adverse Events

In this study, an adverse event (AE) was defined as any untoward medicaloccurrence in a subject or clinical investigation subject administeredBH4, at any dose, whether or not it has a causal relationship with theevent. An AE can therefore be any unfavorable and unintended sign(including an abnormal laboratory finding), symptom, or diseasetemporally associated with the use of BH4, whether or not related toBH4. This definition included intercurrent illnesses or injuries andexacerbation (increase in frequency, severity or specificity) ofpre-existing conditions.

The reporting period for AEs began with the first administration of BH4.The reporting period for serious adverse events (SAEs) began earlier,from the time of the signing of the Informed Consent. SAEs were definedlater in this section. The investigator monitored all AEs untilresolution or, if the AE was determined to be chronic, a cause wasidentified. If an AE remained unresolved at the conclusion of the study,the PI and Medical Monitor made a clinical assessment as to whethercontinued follow-up of the AE was warranted, and documented the results.Assessment of severity was one of the responsibilities of theinvestigator in the evaluation of AEs and SAEs. The investigator wasresponsible for applying his or her clinical judgment to assess thecausal relationship of each AE to BH4.

Serious Adverse Events

A serious adverse event (SAE) was defined as any AE that has at leastone of the following outcomes:

Resulted in death

Was life-threatening, that is, placed the subjects at immediate risk ofdeath from the event as it occurred

This definition did not include a reaction that, had it occurred in amore severe form, might cause death

Required inpatient hospitalization or prolongation of existinghospitalization

Admission of a subject to the hospital as an inpatient as a result of anAE, even if the subject was released on the same day, qualified ashospitalization. An emergency room visit did not constitutehospitalization.

Resulted in persistent or significant disability or incapacity

An event qualified as resulting in a persistent or significantdisability or incapacity if it involved a substantial disruption of thesubject's ability to carry out usual life functions. This definition wasnot intended to include experiences of relatively minor or temporarymedical significance.

Was a congenital anomaly or birth defect, that is, an AE that occurredin the child or fetus of subject exposed to study drug prior toconception or during pregnancy

Was an important medical event that did not meet any of the abovecriteria, but could jeopardize the subject or required medical orsurgical intervention to prevent one of the outcomes listed above.

More than one of the above outcomes could apply to any specific event.

Appropriateness of Measurements

The measures of safety in this study were routine physical examinations,vital signs, adverse event incidence and severity, and clinical andlaboratory procedures.

Drug Concentration Measurements

Blood (plasma) pharmacokinetic (PK) characteristics were assessed aftereach dose of study medication. All subjects remained seated in anupright position for 4 hours post-dose. The blood samples were drawnwithin 30 minutes prior to dose and at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 5.0, 6.0, 8.0, 10.0, 12.0, 18.0, and 24.0 hours post dose.Samples were collected in appropriately labeled 6 mL K₂-EDTA purple topVacutainer® tubes. Blood samples were centrifuged at approximately 3000rpm at 4° C. for 10 minutes. From the resulting plasma, exactly 1 mL wasremoved from each sample using a pipet, and placed into an aliquot tubecontaining 0.1% w/v dithioerythritol. The sample was capped and vortexedfor approximately 10 seconds using a VWR Mini Vortexer at speed 6. Aftercompletion of these steps, the sample was flash frozen in anisopropyl/dry ice bath and placed in a −70° C. freezer pending analysis.

Approximately 80 mL of blood was drawn during each treatment period (5mL per timepoint) for the PK analysis.

Pharmacokinetics:

Pharmacokinetic (PK) analysis of plasma BH4 concentration-time data wasperformed using non-compartmental methods to obtain estimates of thefollowing PK parameters:

-   -   Peak plasma concentration (C_(max)) and time to peak        concentration (T_(max)), obtained directly from the data without        interpolation;    -   λz, the apparent terminal elimination rate constant, determined        by log-linear regression of the terminal plasma concentrations;    -   Area under the plasma concentration-time curve from time zero to        the time of last measurable concentration [AUC(0-t)], calculated        by the linear trapezoidal method;    -   The apparent elimination half-life (t^(1/2)), calculated as        0.693/λz;    -   Area under the plasma concentration-time curve from time 0 to        infinity [AUC(inf)] where AUC(inf)=AUC(0-t)+C_(t)/λz and C_(t)        is the last measurable concentration.

Estimation of Absorption Rate

Subjects were given a 10 mg/kg oral or intravenous dose of BH4, followedby serial measurements of plasma total biopterin concentration todetermine the rate of BH4 absorption from the gastrointestinal tractfrom the area under the plasma total biopterin concentration increase(ΔCp)-time curve (ΔAUC). It was anticipated that a lower dose of BH4 wasrequired when administered intravenously in comparison with BH4administered orally to achieve the same level of bioavailability. Forexample, it may require 10 mg/kg of BH4 given orally to achieve the samelevel of bioavailability as 1 mg/kg BH4 administered intravenously.Because the manner of administration enhanced bioavailability, it mayrequire only 5 mg/kg of BH4 to achieve the same level of bioavailabilityas a 1 mg/kg IV dose of BH4.

The rate of BH4 absorption from the gastrointestinal tract was estimatedfrom the area under the plasma total biopterin concentration increase(ΔCp)-time curve (ΔAUC) after the administration BH4 using the followingformulas:

Estimation from AUC

Absorption rate (%)=(ΔAUC after p.o. dose/ΔAUC after i.v. dose)×(i.v.dose/p.o. dose×100)

Statistical Methods:

Comparison of the pharmacokinetic parameters Cmax, AUC(0-t), andAUC(inf) for BH4 was conducted using an analysis of variance (ANOVA)model with sequence, subject within sequence, treatment, and period asthe classification variables using the natural logarithms of theparameters as the dependent variables. The comparisons of interest werebetween the dissolved and intact tablet in the fasted state and theintact tablet in the fed and fasted states.

The data from all subjects completing at least two study periods wereincluded in the PK statistical analyses. All subjects receiving at leastone dose of study drug were included in the safety analyses.

All PK and associated statistical analyses were done using SAS® forWindows®Version 9.1.3 or higher.

To provide sufficient power to meet the objectives of the study, asample size of approximately 30 subjects, each with 3 treatment periods,was considered adequate to provide estimates of the differencescomparisons of interest. No formal sample size calculation wasconducted.

Results Pharmacokinetics

Intact Versus Dissolved Tablets

Mean plasma concentrations of BH₄ were lower when BH4 was administeredas a dissolved tablet compared to the intact tablet (FIGS. 23 and 24).Mean Cmax was higher for the intact tablet as were mean values forAUC(0-t) and AUC(inf) (FIG. 25). The geometric mean ratios,intact-to-dissolved tablet, ranged from 118% to 121% and the upperlimits of the associated 90% confidence intervals were greater than 125%(FIG. 26), indicating a statistically significant increase in absorptionwhen the intact tablet is administered with a high-calorie, high-fatmealdifference in absorption between dissolved and intact tabletadministration. The median and range for Tmax were essentially the samefor the dissolved and intact tablets (FIG. 25), suggesting that theincrease seen with the intact tablet was in the extent but not the rateof absorption.

Effect of High-Calorie, High-Fat Food on Drug Absorption

As expected, administration of the intact tablet with a standardhigh-fat high-calorie meal resulted in a substantial increase in themean plasma BH₄ concentrations (FIG. 23) and mean values for Cmax,AUC(0-t), and AUC(inf) (FIG. 25). The geometric mean ratios(fed-to-fasted) ranged from 126% to 139% (FIG. 26) and, consequently,the upper limits of the associated 90% confidence intervals were greaterthan 125%, indicating a statistically significant difference in theeffect of food on absorption compared to intact tablets. The median andrange for Tmax were essentially the same under fed and fasted conditions(FIG. 25), suggesting that the increase seen with food was in the extentbut not the rate of absorption of absorption.

Safety:

There were no serious adverse events (SAEs) in this study. Five (5)subjects reported a total of 9 adverse events (AE)s. Eight (8) of these9 AEs were assessed as mild and 1 was assessed as moderate in severity.The most common AE was headache; 1 subject experienced a moderateheadache which was assessed as unrelated to the study drug, and onesubject experienced mild headache on two occasions, both of which wereassessed as possibly related. In all, five events were judged to beunrelated and 4 were judged to be possibly related to the study drug.Study exit assessments, ECG and physical examination evaluations werecompleted with no clinically significant findings.

Conclusions:

Administration of BH4 as an intact tablet resulted in an approximate 20%increase in the extent of absorption compared to a dissolved tablet.

Administration of BH4 as an intact tablet with a high-calorie, high-fatmeal under fed conditions resulted in an approximate 30% increase in theextent of absorption compared to fasted conditions.

No clinically significant issues and safety parameters safety issueswere identified in this study population. There were no AEs consideredserious in this study. Among the 9 AEs reported, all but one, aninstance of headache, was mild, and it was assessed to be unrelated tothe study drug. Instances of fatigue and headache were the only AEswhich were possibly related to the study drug, but and these wereassessed as mild in severity.

Example 4 Formulation Approaches to Enhance Bioavailability of BH4

Two control formulations (BH4 intravenous formulation and BH4 tablet fororal solution) and six test formulations were selected for testing inanimal studies. Each formulation prototype contained 80 mg or 100 mg ofBH4.

BH4 Intravenous Formulations

Table 3 lists the composition of an intravenous formulation. BH4 waspassed through a #20 mesh stainless screen before use while mannitol wasused as received. This formulation was filled as a powder in a bottleand constituted with sterile water for injection prior toadministration. Each bottle contained 100 mg of BH4 and 5 g of mannitolin a clear polyethylene terephthalate copolyester (PETG) bottle with awhite high-density polyethylene (HDPE) screw top closure. Prior toadministration, the formulation was constituted with 100 mL of sterilewater for injection to yield a final concentration of 1 mg/mL. The IVformulation was supplied as a dry powder in a bottle, and each bottlecontained the API and mannitol. The powder was dissolved in sterilewater for injection and filtered prior to administration by IV route.

TABLE 3 Composition of BH4 IV Formulation Ingredients % (w/v) mg/mL BH40.1  1.0 Mannitol (low in 5.0 50.0 endotoxin), USP/Ph. Eur. Sterilewater for qs 100 mL qs 1 mL injection

BH4 Tablet for Oral Solution

Table 4 lists the composition of an oral solution formulation. Ten (10)BH4 tablets (100 mg) were placed into a 125 mL graduated PETG bottlewith a white HDPE closure. Prior to administration, the formulation wasconstituted with 100 mL of sterile water for injection to yield a finalconcentration of 10 mg/mL.

TABLE 4 Composition of BH4 Tablet, 100 mg Ingredients % (w/w) mg/tabletBH4 33.33 99.99 Ascorbic Acid, USP/EP 1.67 5.01 Crospovidone, USP/EP 4.513.5 Dicalcium Phosphate Anhydrous, 2.18 6.54 USP/EP Mannitol (Parteck M200), UPS/EP 57.06 171.18 Riboflavin universal, USP/EP 0.01 0.03 SodiumStearyl Fumarate (PRUV), 1.25 3.75 NF/EP Total 100.00 300.00

Formulation Prototype to Slow Gastro-Intestinal Motility

Table 5 lists the composition of a delayed gastric emptying timeprototype. BH4 was passed through a #20 mesh stainless steel screenbefore use. The Capmul GMO-50 was melted in a 37° C. water bath. BH4 andascorbic acid were weighed and added slowly to the melted Capmul whilestirring vigorously. The solid dispersion was added dropwise into a size#2 capsule using a pipette. Three filled capsules were placed in a 100cc high-density polyethylene (HDPE) bottle with a heat-induction sealclosure.

TABLE 5 Composition of BH4 Delayed Gastric Emptying Time Oral CapsuleFormulation Ingredients % (w/w) mg/capsule BH4 25 80 Glycerylmono/di-oleate (Capmul 65 208 GMO-50) Ascorbic acid fine powder 10 32Total 100 320

Bioadhesive Prototype

Table 6 lists the composition of a bioadhesive prototype. All materials,except for Carbopol 71G, were passed through a #20 mesh stainless steelscreen. All materials were weighed and added to a plastic bag having azip-locking closure, which was then shaken for a few minutes until themixture appeared uniform. The powder was compressed into a tablet usinga ¼″ standard, round, concaved, plain-faced B tooling on a Globe PharmaMTCM-I manual press at 600 psi. Three tablets along with a silica geldesiccant canister were packaged in a 100 cc HDPE with a heat-inductionseal closure.

TABLE 6 Composition of BH4 Bioadhesive Oral Tablet FormulationIngredients % (w/w) mg/tablet BH4 48.5 80.00 Carbopol 71 G 20.0 32.99Polycarbophil (Noveon AA1) 20.0 32.99 Ascorbic acid fine powder 10.016.49 Sodium stearyl fumarate (PRUV) 1.5 2.47 Total 100.0 164.94

Sustained Release Prototype

Table 7 lists the composition of a sustained release prototype tested inthe monkey. All materials, except for Methocel K100M Premium CR, werepassed through a #20 mesh stainless steel screen. All materials wereweighed and added to a plastic bag having zip-locking closure, which wasthen shaken for a few minutes until the mixture appeared uniform. Thepowder was compressed into a tablet using a ¼″ standard, round,concaved, plain-faced B tooling on a Globe Pharma MTCM-I manual press at1200 psi. The tablets along with a Silica gel desiccant canister werepackaged in a 100 HDPE bottle with heat-induction seal closure.

TABLE 7 Composition of BH4 Sustained Release Tablet FormulationIngredients % (w/w) mg/tablet BH4 53.5 80.00 Methocel K100M premium CR35.0 52.34 Ascorbic acid fine powder 10.0 14.95 Sodium stearyl fumarate(PRUV) 1.5 2.24 Total 100.0 149.53

Proton Donor Polymer Prototype

Table 8 lists the composition of a proton donor polymer prototype testedin the monkey. All materials, except for Eudragit L100-55 and KollidonCL, were pre-screened using a #20 mesh stainless steel screen. Allmaterials were weighed and added to a plastic bag having a zip-lockingclosure, which was then shaken for a few minutes until the mixtureappeared uniform. A pre-weighed quantity of powder was filled into asize #2 capsule.

A coating solution was prepared by dissolving Eudragit L100-55 andCarbowax PEG 4600 in Ethyl Alcohol. The Eudragit L100-55 and CarbowaxPEG 4600 were weighed and added to a 125 mL graduated polyethyleneterephthalate copolyester bottle (PETG). The Ethyl Alcohol was added tothe PETG bottle, and it was placed in a 40° C. water bath withsonication until the solution was clear.

The powder-filled capsules were manually dipped into the coatingsolution and allowed to dry at 40° C. for 20 minutes. The dried capsuleswere weighed and then rolled in Syloid FP244 to remove residualtackiness. Three capsules were packaged in a100 cc HDPE bottle with aheat-induction seal closure.

TABLE 8 Composition of BH4 Proton Donor Capsule Formulation IngredientsComposition of Capsule % (w/w) mg/capsule BH4 40.0 80 Eudragit L100-5544.5 89 Crospovidone 4.0 8 (Kollidon CL) Ascorbic acid fine powder 10.020 Sodium stearyl fumarate 1.5 3 (PRUV) Total 100.0 200 IngredientsComposition of Capsule Coating % (w/w) mg/capsule¹ Eudragit L100-55 5.0ND Polyethylene glycol 4600 5.0 ND (Carbowax Sentry) Ethyl alcohol, 200proof 100 mL ND ¹ Following capsule coating and drying in the oven at40° C., the capsule gains about 1 to 3% weight in polymer coating. ND =Not Determined

Floating Delivery System

Table 9 lists the composition of a floating delivery system. Allmaterials, except for Eudragit L100-55, were passed through a #20 meshstainless steel screen. This tablet prototype comprised three layers;the middle layer contained the drug substance, which was sandwichedbetween two water-insoluble outer layers. The inner and outer materialswere weighed and added separately to plastic bags having zip-lockingclosures, which were then shaken until the mixtures appeared uniform.

The two outer layers (12 mg each) and inner layer (14.5 mg) wereweighed. One of the outer layers was added to the press, followed by theinner layer, and then the last outer layer. The layers were compressedinto a tablet using a 3/16″ round, beveled, plain-faced B Tooling on aGlobe Pharma MTCM-I manual press at 200 psi.

A coating solution was prepared by dissolving Ethocel and PEG 4600 in anethyl alcohol and purified water mixture. The ingredients were added toa PETG bottle, which was mixed and placed in a 40° C. water bath withsonication until the solution appeared clear.

The tablets were manually dipped in the coating solution and allowed todry for 20 minutes at 40° C. Each tablet was re-weighed after coating.Seven (7) tablets were placed into each of the size #2 elongatedcapsules. Three capsules were packaged in a 100 cc HDPE bottle with aheat-induction seal closure.

TABLE 9 Composition of BH4 Floating Dosage Formulation % (w/w) mg/tabletIngredients Outer Layers 1 and 3 Eudragit L100-55 49.5 5.94 Stearic acid49.5 5.94 Sodium stearyl fumarate (PRUV) 1.0 0.12 Total 100.0 12.00Ingredients Middle Layer 2 BH4 79.0 11.46 Stearic acid 10.0 1.45Ascorbic acid fine powder 10.0 1.45 Sodium stearyl fumarate 1.0 0.15Total 100.0 14.51 Ingredients 7 tablets in a Capsule % (w/w) mg/capsuleBH4 29.8 80.19 Stearic acid 34.6 93.31 Ascorbic acid fine 3.8 10.15powder Eudragit L100-55 30.8 83.16 Sodium stearyl fumarate 1.0 2.70(PRUV) Total 100.0 269.51 Ingredients Tablet Coating Solution % (w/w)mg/capsule¹ Ethocel Standard 10 FP 5.0 ND Carbowax PEG 4600 5.0 NDEthanol 200 proof 95.0 mL ND Purified Water 5.0 mL ND ¹Following capsulecoating and drying in the oven at 40° C., the capsule gains about 3 to8% weight in polymer coating. ND = Not Determined

Gas Generating Floating Delivery System

Table 10. lists the composition of a gas generating floating deliverysystem. This formulation was composed of a core tablet containing thedrug substance surrounded by a gas-generating outer layer. Allmaterials, except for sodium bicarbonate and Methocel K100M CR, werepre-screened using a #20 mesh stainless steel screen. The inner core andouter layer materials were weighed and added separately to plastic bagshaving zip-locking closures, which were closed and shaken until themixture appeared uniform. The blended powder for the inner core (35 mg)was compressed into a tablet using a ⅛″ round, beveled, plain faced BTooling on a Globe Pharma MTCM-T manual press at 800 psi.

A coating solution was prepared by dissolving using Ethocel and PEG 4600in ethyl alcohol. The inner core tablets were manually dipped in thecoating solution and allowed to dry for 20 minutes at 40° C. The blendedpowder for the outer layer (40 mg) was weighed. One half was added tothe press, followed by the inner core tablet, and then the second halfof the outer layer. The tablet was compressed using a 3/16″ round,beveled, plain-faced B Tooling on a Globe Pharma MTCM-I manual press at800 psi. Four (4) tablets were placed into each size #2 capsule.

TABLE 10 Composition of BH4 Gas Generating Floating Dosage Formulation %(w/w) mg/tablet Ingredients Inner tablet Core BH4 58.3 20.39 Ascorbicacid fine powder 19.4 6.80 HPMC K100MCR 19.4 6.80 Sodium stearylfumarate (PRUV) 2.9 1.02 Total 100 35.01 Ingredients Outer Tablet LayerHPMC K100MCR 46.1 18.46 Citric acid anhydrous 34.2 13.68 Sodiumbicarbonate 17.1 6.84 Sodium stearyl fumarate 2.6 1.03 Total 100 40.01Ingredients Four tablets in a Capsule % (w/w) mg/capsule BH4 27.2 81.55Ascorbic acid fine 9.1 27.18 powder HPMC K100MCR 33.7 101.03 Citric acidanhydrous 18.2 54.70 Sodium bicarbonate 9.1 27.35 Sodium stearylfumarate 2.7 8.18 Total 100 299.99 Ingredients Coating Solution % (w/w)mg/capsule¹ Ethocel Standard 10 FP 5.0 ND ¹Following capsule coating anddrying in the oven at 40° C., the capsule gains weight in polymercoating. ND = Not Determined

Bioadhesive Granule Prototype

Table 11 lists the composition of a bioadhesive granule prototype. Allmaterials, except for Methocel K100M CR, were pre-screened using a#20-mesh stainless steel screen. All materials, except for the sodiumstearyl fumarate (PRUV), were weighed and placed into a size #1granulator bowl (LB Bohle Mini Granulator BMG). The powder was mixed atan impeller speed of 300 rpm and a chopper speed of 2500 rpm for fiveminutes until the mixture appeared uniform. Maintaining the impeller andchopper speeds, 5 mL of ethyl alcohol was added dropwise to the mixtureuntil granules formed. The wet mass was removed from the granulationbowl and screened through an 18-mesh stainless steel screen. Thegranules were collected and placed in a 40° C. oven to dry for one hour.The loss on drying of the granules was determined to be 1.93% after onehour of drying. The granules were weighed and placed into a plastic baghaving a zip-locking closure. Sodium stearyl fumarate (PRUV) was addedto the dried granules in the bag. The bag was closed and shaken untilthe sodium stearyl fumarate (PRUV) appeared evenly distributed among thegranules. The granules were weighed (134 mg). Size 2 elongated capsuleswere filled with portions of the granules alternating with drops ofpartially hydrogenated vegetable oil (350 μL). Three capsules werepackaged in a 100 cc HDPE bottle with a heat-induction seal closure.

TABLE 11 Composition of BH4 Bioadhesive Granule Capsule FormulationIngredients % (w/w) mg/capsule BH4 60 80.00 Methocel K100M CR 19 25.33Carbopol 971 10 13.33 Ascorbic Acid fine power 10 13.33 Sodium StearylFumarate (PRUV) 1 1.33 Pureco HSC-1 oil 350 μL Total 100 133.33

In Vitro Drug Release

In vitro drug release testing from tablets was conducted according tothe USP 27 apparatus 11 specifications using a Distek 2100C DissolutionTester (Distek, Inc., North Brunswick, N.J.), along with an AgilentUV-Visible spectroscopy system (Agilent Technologies, Santa Clara,Calif.). The dissolution medium used for the release testing of BH4 was900 mL of 0.1N HCl. During dissolution testing, the media in each vesselwas maintained at 37°±0.5° C. and agitated at 50 rpm. A sample volume of5 mL was taken at pre-determined time points. To determine theconcentration of BH4 in the samples, 250 μL of each sample was dilutedwith 500 μL of 0.1N HCl and the absorption was measured at 265 nm usinga UV spectrometer (8453 UV-Visible Spectrophotometer, AgilentTechnologies, Santa Clara, Calif.). The data were collected usingChemStation software (Rev. A.09.01[76], Agilent Technologies, SantaClara, Calif.). All dissolution tests were performed in triplicate.

Tablet Buoyancy Testing

The buoyancy of the floating prototype tablets was first determined byplacing the tablets in plastic cups with 25-50 mL of 0.1N HCl. This testdetermined the time necessary for the tablets to float as well as theduration of their floating with no agitation. Those prototypes thatfloated for at least four hours were submitted for dissolution testing.During the dissolution testing, the buoyancy of the tablets wasdetermined using the paddle method at a rotation speed of 50 rpm. Thestate of the tablets was checked visually at various time points.

Disintegration Testing

Disintegration testing was conducted according to the USP-27disintegration test specifications using a Distek 3100 SeriesDisintegration Tester (Distek Inc., North Brunswick, N.J.). Thedisintegration media used was 900 mL of 0.1N HCl or 900 mL of 0.2MPotassium Phosphate pH 5.8. During the disintegration testing the mediain the vessels was maintained at 37°±0.5° C. The tablets and capsuleswere visually inspected for disintegration.

Tablet Hardness Testing

Tablet hardness was determined using a Dr. Schleuniger Pharmatron 8MTablet Hardness Tester (Dr. Schleuniger® Pharmatron Inc., Manchester,N.H.). The tablets were placed into the jaw of the hardness tester, andthe hardness was measured in kiloponds (Kp).

Tablet Thickness

The thickness of the tablets was measured using a Mitutoyo DigimaticIndicator (Mitutoyo Absolute, Dr. Schleuniger Pharmatron Inc.,Manchester, N.H.). The tablets were placed under the thickness gauge andthe value indicated was recorded in millimeters (mm).

Results and Discussion

Several prototypes were developed based on three concepts:gastroretentive, proton donor polymer to change intestinal pH, andsustained release dosage forms. The sections below described theformulation development of each prototype.

BH4 Intravenous Formulation—After sterile water constitution, theresulting solution was isotonic, pH 3.2 and contained 1 mg/mL of BH4,and was suitable for intravenous administration after sterile filtrationthrough a 0.22 micron filter. Stability of the 1 mg/mL solution storedat ambient temperature was analyzed by HPLC every hour for three hours.The aged solution samples were then stored at −20° C. and analyzed byHPLC after 2 weeks. FIG. 27 indicates that the solution was stable atambient temperature for at least 3 hours after constitution and wasstable for at least 2 weeks during storage at −20° C.

BH4 Tablet for Oral Solution

Each bottle was packaged to contain ten (10) BH4 tablets, 100 mg.One-hundred (100) mL of purified water or sterile water for injectionwas added to the contents of each bottle. Following vigorous shaking ofthe bottle, the tablets rapidly disintegrated within 5 minutes. Theresulting solution contained 10 mg/mL of BH4 for oral administration.Not all the ingredients in the tablet were soluble, and although thefinal solution appeared hazy or translucent, the active pharmaceuticalingredient was fully dissolved and the fine particulates were poorlysoluble inactive ingredients.

Formulation Prototype to Slow Gastro-Intestinal Motility

This capsule formulation comprised of BH4 and ascorbic acid dispersed ina semi-solid fatty acid derivative (glyceryl mono/di-oleate, meltingpoint of 86° F. (30° C.)). Glyceryl mono/di-oleate (GMO) was alsoselected because GMO is chemically compatible with BH4. The dissolutionprofile depicted in FIG. 28 showed that over 90% of the drug wasreleased in 2 hours and the dissolution profile remained unchanged afterthe capsules were stored at 40° C. for 57 days.

The drug dispersion in melted GMO, a semi-solid, was filled into hardgelatin capsules manually. The density of the semi-solid is greater than1 g/mL, and it was possible to fill at least 80 mg dose at 25% drugloading in a size #2 capsule. It is expected that a size #0 capsuleshould be able to contain at least 200 mg of drug using the sameformulation. Leakage of fatty acid from the capsule was observed duringstorage at 40° C. Preferably, capsules or softgel capsule formulationswill be banded to avoid leaking of fatty acid during storage.

Bioadhesive Prototype

Many bioadhesives are made of either synthetic or natural polymers. Mostof the current synthetic bioadhesive polymers are either polyacrylicacid or cellulose derivatives. Examples of polyacrylic acid-basedpolymers include but are not limited to carbopol, polycarbophil,polyacrylic acid (PAAc), etc. Cellulosics include but are not limited tohydroxypropyl cellulose and hydroxypropylmethyl cellulose (HPMC). Twobioadhesive prototypes were developed for testing in animal studies. Thefirst prototype was a bioadhesive tablet formulation and the second acapsule containing bioadhesive granules.

Polycarbophil and carbomer polymers were selected for the development ofthe first bioadhesive tablet prototype. Carbopol 71 G is a granular formof carbomer and has good powder flow properties. All the batches of thefabricated tablets were of good quality with acceptable drug content(evident by close to 100% drug release in dissolution profiles) andacceptable hardness. Table 12 lists the representative tablet weight,thickness, and hardness of the bioadhesive prototype containing carbomerand polycarbophil.

TABLE 12 Representative Tablet Weight, Thickness and Hardness forBioadhesive Prototype containing Carbomer and Polycarbophil Tablet LotCompression Weight Number Pressure (psi) (mg) Thickness (mm) Hardness(Kp) 11210-83 600 165.4 5.24 10.5 11229-4 600 166.7 5.64 10.3 11229-4800 164.1 5.27 14.4 11229-4 1000 164.9 5.12 18

HPMC and carbomer polymers were used for the development of the secondbioadhesive granules. HPMC was selected because it is used aslow-density hydrocolloid system and controlled drug release independentof pH. Granules were selected over tablet to increase the chance ofbioadhesion by increasing the surface area of the dosage form. Tofacilitate the separation of the granules-filled capsule in dissolutionmedium, the granules were coated partially with hydrogenated oil.Without the oil coating, the granules hydrated and formed acapsule-shaped matrix without disintegrating into individual granules.

The release profiles of the two bioadhesive prototypes (tablet andgranules) are shown in FIG. 29, which shows that the release profile ofthe tablet was longer than the granules. Drug release was about 90% infour hours and 95% in one hour for the tablet and granules bioadhesivedosage forms, respectively. Upon storage at 40° C. and ambient humidityfor one month without moisture protection (no heat induction seal), thetablet prototype exhibited a slowdown in drug dissolution (FIG. 29). Forprototypes containing carbomer, moisture protection precaution should betaken to protect the tablet from possibly hydrating prematurely.Sustained Release Prototype

Hydroxypropylmethylcellulose (HPMC) is used as a hydrophilic vehicle forthe preparation of oral controlled drug delivery systems (Colombo, Adv.Drug Deliv. Rev., 1993, 11, 37). HPMC matrices are known to control therelease of a variety of drugs (Chattaraj, et al. Drug Develop. Ind.Pharm., 1996, 22, 555; Pabon, et al., Drug Develop. Ind. Pharm., 1992,18, 2163; Lee, et al., Drug Develop. Ind. Pharm., 1999, 25, 493; Basak,et al., Indian J. Pharm. Sci., 2004, 66, 827; Rajabi-Siabhoomi, et al.,J. Pharm. Pharmacol., 1992, 44, 1062). Various viscosity grades of HPMC(K4M, K15M and K100 M) to control the release of BH4 were evaluated inthis study. The dissolution profiles of tablets made with various gradesof HPMC are shown in FIG. 30. Drug release profiles were similar at 20%HPMC regardless of viscosity grade; over 80% of the drug was released in2 hours. When HPMC polymer was exposed to aqueous medium, it underwentrapid hydration and chain relaxation to form gel layer (Naruhashi, etal., Pharm Res. 2003, 19:1415-1421). The HPMC at 20% may not form asubstantial gel barrier layer to slow the release of BH4 significantly.

The dissolution profiles of tablets produced with varying concentrations(20% to 40%) of a high viscosity grade of HPMC (Methocel K100M CR) arepresented in FIG. 30. A tablet containing 35% to 40% Methocel K100M CRwas found to slow drug release for up to four hours whereas 20% HPMCreleased drug in two hours (FIG. 31). A tablet containing 35% HPMC(Methocel K100M) was selected as the prototype for testing in animalstudies because it contained the least amount of HPMC required to slowthe drug release for up to four hours. As such, the tablets were of goodquality with acceptable drug content as evident by close to 100% drugrelease in dissolution profiles.

Proton Donor Polymer Prototype

To increase the oral absorption of BH4, one approach is to stabilize thedrug by decreasing the pH of the proximal small intestine. To manipulateintestinal luminal pH, Eudragit L100-55, a proton-releasing polymercommonly used for enteric coating, was selected. This polymer is notsoluble under acidic conditions, and it becomes soluble and releasesprotons under weakly acidic (pH >5.5) to alkaline condition due to itscarboxyl groups, thereby controlling the intestinal luminal pH to beacidic. Naruhashi, et al. (2003) found that pH in the lumen wasdecreased in a Eudragit L100-55 concentration-dependent manner and theabsorption of cefadroxil and cefixime from the ileal loop was increasedin the presence of the acidic polymer (Nozawa, et al., J. Pharm Sci.2003, 92 (11), 2208-2216). Nozawa, et al (2003) showed than Eudragitdecreased the pH in the intestinal loops, and increased thedisappearance of both cefadroxil and cefixime from the loops.

Powder formulations containing BH4 and Eudragit L100-55 as shown inTable 8 were compressed into tablets and filled into capsules. Thetablet formulation released about 27% drug in one hour in simulatedgastric fluid (SGF) during dissolution testing. However, duringdisintegration testing, the tablet remained intact in SGF and pH 5.8phosphate buffer (PB) for at least 2 hours. Even in the presence of asuper-disintegrant (crospovidone or croscarmellose), the tablet failedto disintegrate. It is possible that the drug may be acidifying theEudragit, creating a low micro pH environment such that the polymerremained unionized and insoluble.

The powder filled capsule drug-Eudragit formulation disintegratedrapidly in SGF. To target proton release in the proximal intestine, anenteric coat was applied to the capsule. Following capsule coating anddrying in the oven at 40° C., the capsule gained about 1 to 3% weight inpolymer coating. When tested using the USP dissolution apparatus 11(paddle), dissolution medium 0.1 N HCl maintained at 37° C. at arotational speed of 50 rpm, the coated capsule released about 25% ofdrug in one hour. Following 1 hour of acid (0.1 N HCl) pre-treatment,the coated capsule was placed in a USP disintegration Apparatus with 500mL of pH 5.8 phosphate buffer maintained at 37° C., the coated capsuledisintegrated in about 1 hour. The enteric-coated capsule prototype wasselected over the tablet or the uncoated capsule because theenteric-coated capsule was more likely to deliver proton-releasingpolymer to the target site.

Floating Delivery System

Two floating delivery systems were developed. The first prototype was afloating multiple unit dosage form; the purpose of this dosage form wasto increase the chance that one of the units will remain in the gastricregion and hence prolong the gastric residence time of drugs. Thisdosage form consisted of seven triple layer tablets in a capsule; themiddle layer contained the drug substance, which was sandwiched betweentwo water-insoluble outer layers (FIG. 32). The outer layers containedstearic acid, a hydrophobic and water-insoluble fatty acid, whichprovided the necessary buoyancy to the floating tablet. Each tablet wasmanually coated with an alcoholic solution of ethylcellulose andpolyethylene glycol MW 4600 (PEG). Ethylcellulose formed a waterinsoluble film around the tablet and PEG, which acted as a pore former,modulated the release rate. The dissolution profiles of tablets coatedwith ethylcellulose and various concentrations (20% to 40%) of PEGsolutions are presented in FIG. 33. It was noted that the coated triplelayer tablet achieved close to zero-order release kinetics. As expected,the drug dissolution rate increased as the concentration of PEGincreased. The tablets floated in simulated gastric medium for at leastfour hours during dissolution studies. Table 9 shows the composition ofthe formulation tested in animal studies.

The second prototype was a gas-generating dosage form. It was formulatedin such a way that when it came in contact with acidic gastric contents,carbon dioxide was liberated and got entrapped in the swollenhydrocolloids, which provided buoyancy to the dosage form (FIG. 33).This formulation floated in simulated gastric medium for at least fourhours during dissolution studies. However, for such a system to workconsistently, the tablets have to be produced in a low humidityenvironment to prevent premature acid and base reaction. There could bepotential interaction between BH4 and sodium bicarbonate in the tabletduring storage. For these reasons, this dosage form was not tested inanimal studies.

Six prototype test formulations that incorporated various formulationapproaches including proton donor polymer to decrease intestinal pH,gastroretentive dosage forms, and sustained released formulations, weredeveloped for animal bioavailability studies.

Example 5 Bioavailability of Novel BH4 Formulations

The objective of this study was to enhance the absorption of BH4 bydeveloping dosage forms that increase the residence time of the drug inthe gastrointestinal (GI) tract.

Methods: Three healthy cynomolgus monkeys weighing 3-4 kg were used inopen, 8-period non-crossover study to determine the bioavailability ofseven formulations compared to a control dissolved BH4 formulation.After an overnight fast, the monkeys received, on separate occasions, asingle dose of 80 mg of the same novel formulation orally orintravenously with an interval of at least a 1 week washout periodbetween the various novel formulations studied. For intravenousadministration, blood samples were collected before dosing and then at5, 15 and 30 min and 1.0, 2.0, 4.0, 6.0, 8.0, 12 and 24 hr post dose.For oral administration, blood samples were taken before dosing and then15 and 30 min and 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12, and 24 hr followingeach dose. Following separation of the plasma by centrifugation, 200-μLaliquots of each sample were promptly transferred into individual tubescontaining 0.1% DTE and frozen at −70° C. until ready for assay fortotal L-biopterin.

Study Formulations: The formulations administered are found in Table 13.Three of the formulations were conceptually designed to begastroretentive via bioadhesive or floating mechanisms to increase GIresidence time (carbomer-based, multi-particulate floating granules andbioadhesive granules). Other concepts were based on slowing GI motilityto increase residence time of the formulation (glyceryl mono-oleate),reducing the pH of the small intestine and thereby enhancing BH4chemical stability to enable absorption of intact drug (proton pump) orsustained delivery formulation to ascertain whether it will enhanceabsorption.

TABLE 13 Dosage Phase Prototype Form Concept Ingredients Phase I IV IVControl BH4, D(−)-Mannitol Formulation solution, 1 mg/mL Phase II KuvanOral Control BH4 tablets Tablets for Solution, manufactured Solution 10mg/ml by Lyne (Lot# 140651) Phase III Glycerol Capsule, Slow GI BH4,Capmul Mono 80 mg motility GMO-50, Ascorbic Oleate Acid Phase IVCarbomer Tablet, Gastro- BH4, Carbopol Prototype 80 mg retentive, 71G,Noveon AA1, Bioadhesive Ascorbic Acid, PRUV Phase V HPMC Tablet,Sustained BH4, Methocel prototype 80 mg release K100M Premium CR,Ascorbic Acid, PRUV Phase VI Eudragit Capsule, Proton BH4, EudragitPrototype 80 mg donor L100-55, Ascorbic polymer to Acid, Kollidon lowerGI pH CL, PRUV, Coating (Eudragit L100-55, Carbowax PEG 4600, EthylAlcohol 200 proof) Phase Multi- Multiple Gastro- Inner Layer (BH4, VIIfloating tablets in retentive, Ascorbic Acid, units capsule, floatingStearic Acid, 80 mg PRUV), Outer Layer (Stearic Acid, Eudragit L100-55,PRUV), Coating (Ethocel Standard 10FP, Carbowax PEG 4600, 95% Ethanol)Phase Bioadhesive Granules Gastro- Intergranular (BH4, VIII Granules incapsule, retentive, Methocel K100M 80 mg Bioadhesive Premium CR,Carbopol 971, Ascorbic Acid), Extragranular (PRUV, Pureco HSC-1 oil)

Plasma Assay for Biopterin: BH4 concentrations in plasma were determinedby using a validated, specific, reversed-phase LC/MS/MS method. Thestandard curve was linear over the concentration range of 50 ng/mL to2500 ng/mL. The lower limit of quantitation for L-biopterin was 50 ng/mLwith intraday precision shown by coefficients of variation less than 5%.L-biopterin is stable in frozen monkey plasma stabilized with 0.1% DTEat −70° C. until assayed. BH4 concentrations were calculated from thedetermined L-biopterin concentrations.

Pharmacokinetic and Statistical Analysis: Pharmacokinetic parameterswere determined for plasma BH4 following the administration of the oraland intravenous formulations. The pharmacokinetic parameters areprovided in Table 14.

TABLE 14 Phase, AUC_(last) AUC_(∞) C_(max) C_(last) ^(a) T_(max) t_(1/2)Formulation (ng-hr/mL) (ng-hr/mL) (ng/mL) (ng/mL) (hr) (hr) 2, dissolved641 (88)  805 (36)  93.6 (31.3) 9.60 (2.20) 2.33 (0.58) 11.7 (2.1) tablet 3, glyceryl 716 (154) 858 (317) 133 (83)  6.47 (3.60) 2.00 (0)  12.1 (10.3) mono-oleate 4,  593 (50.6) 648 (114) 108 (15)  4.46 (3.36)2.67 (0.58) 6.89 (3.51) bioahestive polymer 5, sustained 355 (134) 472(36)  86.0 (43.1) 12.9 (12.4) 3.33 (0.58) 5.30 (1.73) release 6, proton 276 (49.8) 282 (49)  68.3 (25.3) 2.97 (0.71) 3.33 (0.58) 1.59 (0.74)donor 7, floating 304 (78)  b 59.9 (31.8) 5.90 (0.94) 4.00 (2.00) bdosage form 8, 292 (79)   366 (40.6) 42.5 (12.6) 5.11 (2.43) 3.0 (0)  15.3 (8.2)  bioadhesive granulations

Results

The objective of this study was to identify formulations that enhancethe bioavailability BH4 compared to the control dissolved tabletformulation. The mean plasma BH4 concentration-time profiles of thevarious dosage forms and the control formulation following the oraladministration of BH4 are shown in FIG. 35, and the BH4 pharmacokineticparameters derived from plasma drug concentration-time profiles aregiven in Table 14. The control formulation (phase 2) is the dissolvedtablet.

As shown in FIG. 35, the glyceryl mono-oleate formulation provided thehighest AUC_(last) and AUC_(∞) which are 716 ng-hr/mL and 858 ng-hr/mLrespectively. The control dissolved BH4 tablet formulation exhibitedAUC_(last) and AUC_(∞) which are 641 ng-hr/mL and 805 ng-hr/mLrespectively (Table 14). The rank order of the formulations from themost to the least bioavailable is: glyceryl mono-oleate >dissolvedtablet >bioadhesive polymer tablet >sustained release tablet >floatingdosage forms >bioadhesive granulations capsule product >proton donorcapsule product.

Example 6 Preparation of Intravenous Formulation of TetrahydrobiopterinPreformulation Stability Evaluation

In general, the objective of this study was to evaluate the stability ofBH4 in buffer solutions ranging in pH from pH 1 to 7 (See Table 15) andin the presence and absence of antioxidants and with or without inertgas in the reaction solutions (See Table 16).

TABLE 15 Components and Composition of Buffer Solutions to be used forBH4 Preformulation Stability Studies Components Quantities pH 1.2 Buffer(0.1N HCl) Concentrated HCl (12N) 8.33 mL Sodium Chloride 2.92 gDistilled/Deionized Water qs 1000 mL pH 2.1 Buffer (0.01N HCl) pH 1.2(0.1N HCl) Buffer 100 mL Sodium Chloride 7.79 g Distilled/DeionizedWater qs 1000 mL pH 3 Buffer Phosphoric Acid, 15M, 85% .347 mL SodiumMonobasic Phosphate, anhydrous (NaH₂PO₄) 6.17 g Sodium Chloride 6.16 gDistilled/Deionized Water qs 1000 mL pH 4 Buffer Acetic Acid, Glacial,100% 2.38 mL Sodium Acetate, Trihydrate 1.29 g Sodium Chloride 8.22 gDistilled/Deionized Water qs 1000 mL pH 5 Buffer Acetic Acid, Glacial,100% .87 mL Sodium Acetate, Trihydrate 4.78 g Sodium Chloride 6.72 gDistilled/Deionized Water qs 1000 mL pH 6 Buffer4-Morpholineethanesulfonic (MES) Acid Monohydrate 4.99 g MES Sodium Salt5.75 g Sodium Chloride 7.23 g Distilled/Deionized Water qs 1000 mL pH 7Buffer Sodium Monobasic Phosphate, Monohydrate (NaH₂PO₄) 2.56 g SodiumDibasic Phosphate, anhydrous (Na₂HPO₄) 4.44 g Sodium Chloride 2.18 gDistilled/Deionized Water qs 1000 mL

TABLE 16 Composition of Buffer Solutions for Stability StudiesContaining BH4 With or Without Antioxidant and whether Subjected to GasSparging or Not Study Group Number 5 2 3 Buffer + 6 1 Buffer + Buffer +Argon Buffer + Oxygen Buffer Ascorbic L-Cysteine Sparging Sparging pHStudy Acid Study Study Study Study 1 1 mg/mL 1 mg/mL BH4 1 mg/mL BH4 1mg/mL BH4 1 mg/mL BH4 BH4 in pH and 1 mg/mL and 1 mg/mL in pH 1.2 in pH1.2 1.2 Buffer Ascorbic Acid L-Cysteine in Buffer and Buffer and in pH1.2 pH 1.2 Buffer Argon-Sparged Oxygen- Buffer and Argon Sparged and O₂blanket-Sealed blanket-Sealed 2 1 mg/mL 1 mg/mL BH4 1 mg/mL BH4 1 mg/mLBH4 1 mg/mL BH4 BII4 in pII and 1 mg/mL and 1 mg/mL in pII 2.1 in pII2.1 2.1 Buffer Ascorbic Acid L-Cysteine in Buffer and Buffer and in pH2.1 pH 2.1 Buffer Argon-Sparged Oxygen- Buffer and Argon Sparged and O₂blanket-Sealed blanket-Sealed 3 1 mg/mL 1 mg/mL BH4 1 mg/mL BH4 1 mg/mLBH4 1 mg/mL BH4 BH4 in pH 3 and 1 mg/mL and 1 mg/mL in pH 3 Buffer in pH3 Buffer Buffer Ascorbic Acid L-Cysteine in and Argon- and Oxygen- in pH3 Buffer pH 3 Buffer Sparged and Sparged and O₂ Argon blanket-blanket-Sealed Sealed 4 1 mg/mL 1 mg/mL BH4 1 mg/mL BH4 1 mg/mL BH4 1mg/mL BH4 BH4 in pH 4 and 1 mg/mL and 1 mg/mL in pH 4 Buffer in pH 4Buffer Buffer Ascorbic Acid Ascorbic Acid and Argon- and Oxygen- in pH 4Buffer in pH 4 Buffer Sparged and Sparged and O₂ Argon blanket-blanket-Sealed Sealed 5 1 mg/mL 1 mg/mL BH4 1 mg/mL BH4 1 mg/mL BH4 1mg/mL BH4 BH4 in pH 5 and 1 mg/mL and 1 mg/mL in pH 5 Buffer in pH 5Buffer Buffer Ascorbic Acid L-Cysteine in and Argon- and Oxygen- in pH 5Buffer pH 5 Buffer Sparged and Sparged and O₂ Argon blanket-blanket-Sealed Sealed 6 1 mg/mL 1 mg/mL BH4 1 mg/mL BH4 1 mg/mL BH4 1mg/mL BH4 BH4 in pH 6 and 1 mg/mL and 1 mg/mL in pH 6 Buffer in pH 6Buffer Buffer Ascorbic Acid L-Cysteine in and Argon- and Oxygen- in pH 6Buffer pH 6 Buffer Sparged and Sparged and O₂ Argon blanket-blanket-Sealed Sealed 7 1 mg/mL 1 mg/mL BH4 1 mg/mL BH4 1 mg/mL BH4 1mg/mL BH4 BH4 in pH 7 and 1 mg/mL and 1 mg/mL in pH 7 Buffer in pH 7Buffer Buffer Ascorbic Acid L-Cysteine in and Argon- and Oxygen- in pH 7Buffer pH 7 Buffer Sparged and Sparged and O₂ Argon blanket-blanket-Sealed Sealed

More specifically, the influence of combining two antioxidants in thepresence or absence of inert gas was evaluated at pH 4 to support theformulation of a liquid product, and at a pH 7 to ascertain thecontribution of instability at physiologic pH to the low bioavailabilityof the compound in monkeys and humans (See Tables 17 and 18). Thestability of BH4 is expected to be temperature-dependent. Therefore, thecompound stability was evaluated at 2-8° C., 25° C., 30° C. and 37° C.to support the determination of predictive long-term shelf lives for thecompound at different temperatures. Determination of the stability ofthe compound at the physiologic temperature of 37° C. provides data tosupport the estimation of the stability lifetime of a formulated oraldosage form in the absorptive regions of the GI tract.

TABLE 17 Composition of Buffer Solutions for the pH 4 Stability Study ofBH4 pH 4 pH 4 Buffer + Ascorbic Acid + Buffer + Ascorbic Acid +L-Cysteine Study L-Cysteine + Argon Sparge Study 1 mg/mL BH4 and 1 mg/mL1 mg/mL BH4 + 1 mg/mL Ascorbic Acid and Ascorbic Acid + I mg/mL I mg/mLL-Cysteine in pH L-Cysteine in pH 4 Buffer 4 Buffer and Argon-Spargedand Argon blanket-Sealed

TABLE 18 Composition of Buffer Solutions for the pH 7 Stability Study ofBH4 pH 7 pH 7 Buffer + Ascorbic Acid + Buffer + Ascorbic Acid +L-Cysteine Study L-Cysteine + Argon Sparge Study 1 mg/mL BH4 and 1 mg/mL1 mg/mL BH4 + 1 mg/mL Ascorbic Acid Ascorbic Acid + I and I mg/mLL-Cysteine mg/mL L-Cysteine in pH in pH 7 Buffer 7 Buffer and Argon-Sparged and Argon blanket-Sealed

Proposed sampling times for studies to be conducted in various buffersolutions were estimated by comparing the half-life of a single study atpH 3.1 with data obtained by Davis, et al. (1988; Eur. J. Biochem. 173,345-351, (1988)), in pH 6.8 Tris and phosphate buffers. The stabilitystudy of a pH 3.1 solution yielded an estimated t_(1/2) of 17769 min(12.3 days) and the work of Davis et al yielded a t_(1/2) of 10 min inphosphate pH 6.8 buffer and 14 min in pH 6.8 Tris buffer. These twostudies suggest an order of magnitude reduction in half-life (i.e. anorder of magnitude increase in reactivity) of BH4 for every one-foldincrease in pH (see Table 19). Based on this approximation, pH 1.2 to pH3 solutions were sampled weekly initially and sampling time correctionswere made if necessary after the first 2 data points were collected. Theestimated sampling times at 25° C. are provided in Table 19.

TABLE 19 Suggested Sampling Times at Various pH Based on MeasuredHalf-life of BH4 and Theoretical Half-Lives Derived from Them MeasuredEstimated t_(1/2) Based on t_(1/2) Initially Suggested^(c) pH t_(1/2)(Min) Obtained at pH 3 (Min)^(a) Sampling Time 1.0 — 776900.0 (1234days) Every 7 days 2.0 — 177690.0 (123.4 days) Every 7 days 3.0 17769.0 17769.0 (12.34 days) Every 96 hours (12.34 days) 4.0 —  1776.9 (1.23days) Every 12 hours 5.0 —   177.7 (0.12 days) Every ½ Hour 6.0 —   17.7(0.01 days) Every 5 minutes^(d) 6.8^(b) 10 (Phosphate) 14 (Tris) 7.0 —1.8 Every ½ minutes^(d) ^(a)Estimated t_(1/2) is based on changing by anorder of magnitude, the half-life obtained at pH 3.0 for every one-foldchange in pH. pH < 3 are increased upwards while pH > 3 are decreaseddownwards by an order of magnitude in a stepwise fashion to roughlymatch the pH 6.8 data obtained by Davis et al.. ^(b)Data obtained fromDavis, et al. 1988; Eur. J. Biochem., 173, 345-351, (1988) ^(c)Samplingcan be modified ^(d)Reaction solutions are sampled and quenched as fastas possible and require a stopwatch and 2 people, one sampling/quenchingand the other accurately recording the time in a notebook in minutesand/or seconds

Studies were conducted in pH 1-7 buffer solutions and at 5° C., 25° C.,30° C. and 37° C. Although these studies were conducted innon-hermetically sealed containers, anti-oxidants alone (ascorbic acidor L-cysteine) or combined together (ascorbic acid+L-cysteine) reducedthe rate of loss or degradation of BH4 (see FIG. 36 and FIG. 37).Sparging a solution containing both ascorbic acid and L-cysteinesubstantially enhanced the stability of BH4.

The rate of degradation of BH4 is concentration-dependent (see FIG. 38).Therefore high dose, highly concentrated formulations of BH4 were shownto require lower concentration of stabilizers for synergisticstabilization of the formulations.

This results demonstrate that formulation of long shelf-life, stable,liquid formulations can be produced according to the methods andcompositions described herein, including sterile injectable liquids,oral liquids, and lyophilized and sterile powders for constitutionformulations.

Example 7 Liquid and Lyophilized Formulations of Tetrahydrobiopterin forOral and Parenteral Use Example Compositions of Formulations

TABLE 20 Specific formulation buffered at pH 4 having ascorbic acid asstabilizer % Amount Weight/ Components (mg) Volume Function BH4 1.000.10 Active substance Ascorbic Acid 10.00 1.00 Antioxidant Citric Acid6.56 0.66 Buffering agent Sodium Citrate, Dihydrate 5.53 0.55 Bufferingagent Water for Injection qs 1.00 mL 1.00 mL Diluent

TABLE 21 Formulation buffered at pH 4.0 containing a combination of twostabilizers: ascorbic acid and sodium metabisulfite % Amount Weight/Components (mg) Volume Function BH4 1.00 0.10 Active substance AscorbicAcid 2.50 0.25 Antioxidant Sodium Metabisulfite 2.50 0.25 AntioxidantCitric Acid 6.56 0.66 Buffering agent Sodium Citrate, Dihydrate 5.530.55 Buffering agent Water for Injection qs 1.00 mL 1.00 mL Diluent

TABLE 22 Formulation buffered at pH 4.0 containing a combination ofthree stabilizers: L-cysteine, ascorbic acid and sodium metabisulfite %Amount Weight/ Components (mg) Volume Function BH4 1.00 0.10 Activesubstance Ascorbic Acid 2.00 0.20 Antioxidant Sodium Metabisulfite 2.000.20 Antioxidant L-Cysteine 4.00 0.40 Antioxidant Citric Acid 6.56 0.66Buffering agent Sodium Citrate, Dihydrate 5.53 0.55 Buffering agentWater for Injection qs 1.00 mL 1.00 mL Diluent

TABLE 23 Formulation buffered at pH 7.0 containing only ascorbic acidonly as stabilizer % Amount Weight/ Components (mg) Volume Function BH410.00 1.00 Active substance Ascorbic Acid 50.00 5.00 Antioxidant SodiumMonobasic 10.24 0.10 Buffering agent Phosphate, Monohydrate SodiumDibasic Phosphate 17.76 0.18 Buffering agent Water for Injection qs 1.00mL 1.00 mL Diluent

TABLE 24 Formulation buffered at pH 7.0 containing ascorbic acid sodiummetabisulfite as stabilizers % Amount Weight/ Components (mg) VolumeFunction BH4 10.00 1.00 Active substance Ascorbic Acid 20.00 2.00Antioxidant Sodium Metabisulfite 15.00 1.50 Antioxidant Sodium Monobasic10.24 0.26 Buffering agent Phosphate, Monohydrate Sodium DibasicPhosphate 17.76 0.44 Buffering agent Water for Injection qs 1.00 mL 1.00mL Diluent

TABLE 25 Formulation buffered at pH 7.0 containing ascorbic, sodiummetabisulfite and L-Cysteine as stabilizers % Amount Weight/ Components(mg) Volume Function BH4 10.00 1.00 Active substance Ascorbic Acid 20.002.00 Antioxidant Sodium Metabisulfite 15.00 1.50 Antioxidant L-Cysteine10.00 1.00 Antioxidant Sodium Monobasic 10.24 0.26 Buffering agentPhosphate, Monohydrate Sodium Dibasic Phosphate 17.76 0.44 Bufferingagent Water for Injection qs 1.00 mL 1.00 mL Diluent

High Dose Liquid Formulations

TABLE 26 Formulation buffered at pH 6.0 containing ascorbic acid only asstabilizer % Amount Weight/ Components (mg) Volume Function BH4 50.000.10 Active substance Ascorbic Acid 7.50 0.75 Antioxidant Citric Acid5.30 0.53 Buffering agent Sodium Citrate, Dihydrate 51.4 5.14 Bufferingagent Water for Injection qs 1.00 mL 1.00 mL Diluent

TABLE 27 Formulation buffered at pH 6.0 containing a combination of twostabilizers: ascorbic acid and sodium metabisulfite % Amount Weight/Components (mg) Volume Function BH4 50.00 5.00 Active substance AscorbicAcid 2.50 0.25 Antioxidant Sodium Metabisulfite 2.50 0.25 AntioxidantCitric Acid 5.30 0.53 Buffering agent Sodium Citrate, Dihydrate 51.45.14 Buffering agent Water for Injection qs 1.00 mL 1.00 mL Diluent

TABLE 28 Formulation buffered at pH 6.0 containing a combination ofthree stabilizers: L-cysteine, ascorbic acid and sodium metabisulfite %Amount Weight/ Components (mg) Volume Function BH4 50.00 0.10 Activesubstance Ascorbic Acid 2.00 0.20 Antioxidant Sodium Metabisulfite 2.000.20 Antioxidant L-Cysteine 1.00 0.10 Antioxidant Citric Acid 5.30 0.53Buffering agent Sodium Citrate, Dihydrate 51.4 5.14 Buffering agentWater for Injection qs 1.00 mL 1.00 mL Diluent

TABLE 29 Oral formulation buffered at pH 3.0 citrate buffer andcontaining ascorbic acid only as stabilizer % Amount Weight/ Components(mg) Volume Function BH4 10.00 1.00 Active substance Ascorbic Acid 20.002.00 Antioxidant Sucrose 200.00 20.00 Sweetener Orange Flavor 1.00 0.10Flavoring agent Citric Acid 8.98 0.90 Buffering agent Sodium Citrate,Dihydrate 2.13 0.21 Buffering agent Water for Injection qs 1.00 mL 1.00mL Diluent

TABLE 30 Oral formulation buffered at pH 3.5 tartrate buffer andcontaining ascorbic acid and sodium metabisulfite as stabilizers %Amount Weight/ Components (mg) Volume Function BH4 10.00 1.00 Activesubstance Ascorbic Acid 20.00 2.00 Antioxidant Sodium Metabisulfite 5.000.50 Antioxidant Sucrose 200.00 20.00 Sweetener Grape Flavor 1.00 0.10Flavoring agent Tartaric Acid 1.34 0.13 Buffering agent Sodium TartrateDibasic 8.39 0.84 Buffering agent Dihydrate Water for Injection qs 1.00mL 1.00 mL Diluent

TABLE 31 Oral formulation buffered at pH 3.5 in malic acid based bufferand containing ascorbic acid and sodium metabisulfite as stabilizers %Amount Weight/ Components (mg) Volume Function BH4 10.00 1.00 Activesubstance Ascorbic Acid 20.00 2.00 Antioxidant Sodium Metabisulfite15.00 1.50 Antioxidant Sucrose 200.00 20.00 Sweetener Apple Flavor 1.000.10 Flavoring agent Malic Acid 3.07 0.31 Buffering agent Sodium MalateDibasic 4.91 0.49 Buffering agent Water for Injection qs 1.00 mL 1.00 mLDiluent

The foregoing formulated or compounded solutions are optionally spargedwith an inert gas (e.g., argon or nitrogen) or carbon dioxide in thecompounding tank and primary containers preferably are sealed in ablanket of inert gas or carbon dioxide to remove oxygen from thecontainer headspace. The formulations can be scaled up to any volume bymultiplying the component amounts by an appropriate scale up factor.

Example 8 LC/MS/MS Determination of Tetrahydrobiopterin (BH4) in HumanPlasma by Measuring L-Biopterin Concentration Upon Oxidation Under BasicConditions

Tetrahydrobiopterin (BH4) is a small molecule therapeutic for thetreatment of patients with phenylketonuria (PKU). It is important tohave an accurate and specific method to measure BH4 concentrations inhuman plasma. However, it is a challenge to quantify BH4 in human plasmabecause of its low endogenous concentration and instability. Under basicconditions, BH4 is oxidized into dihydrobiopterin (BH2) and ultimatelyL-Biopterin. Furthermore, the oxidation conversion ratio of BH4 toL-Biopterin is nearly constant up to 23 weeks. Therefore, by measuringL-Biopterin concentration upon oxidation under basic condition, andapplying a molar conversion ratio, we can reliably determine the BH4concentrations in human plasma.

Published methods are based on the classical method developed byFukushima and Nixon (Anal. Biochem., 102, 176-188 (1980)) using HPLCwith fluorescence detection. In the LC/MS/MS method, the human plasmasample was stabilized with antioxidant, spiked with an internal standard(IS) solution and basified with sodium hydroxide solution, then oxidizedwith iodine solution. Upon incubation in dark at room temperature,ascorbic acid is added to reduce the excess iodine. Oxidized sampleswere extracted by protein precipitation. L-Biopterin in thereconstituted extracts was analyzed by using reversed-phase HPLC withTurbo Ion Spray® MS/MS detection. Negative ions for L-Biopterin weremonitored in MRM mode. Drug-to-IS peak area ratios for the standardswere used to create a linear calibration curve using 1/x² weightedleast-squares regression analysis.

The oxidation conversion ratio of BH4 to L-Biopterin was evaluated atmultiple time-points: 0, 1, 2, 4, 8, 12 and 23 weeks, and foundconsistent in all the tested time-points with a nominal molar conversionratio of 47.3% determined from the first three consecutive time-points.The difference between the conversion ratio at other time-points and thenominal value ranges from −2.3 to 6.3%. The LC/MS/MS method wasvalidated to quantify L-Biopterin in K₂ EDTA human plasma in the linearcalibration range of 5 to 1000 ng/mL (equivalent to 11 to 2114 ng/mL forBH4). The assay precision and accuracy was evaluated with qualitycontrol samples (QCs) and the results showed intraday precision between4.7 to 14.5% CV; intraday accuracy between −7.1 to 7.4% nominal values;and interday precision and accuracy of 7.4 to 16.4% CV and −8.3 to 3.7%nominal values, respectively. The mean extraction recovery forL-Biopterin was 65.3%. In K₂ EDTA human plasma, L-Biopterin was found tobe stable at room temperature for at least 4 hours and after 4 freezethaw cycles, and at −70° C. for at least 275 days.

Example 9 Determination of BH4/BH2/B Using HPLC with Electrochemical andFluorescence Detection

A study was performed to develop a method of determiningtetrahydrobiopterin (BH4), dihydrobiopterin (BH2) and biopterin (B)concentrations in human plasma using reverse phase high performanceliquid chromatography(HPLC) with fluorescence detection (FD) andelectrochemical detection (ECD). The method is based on Cai, et al.(Cardiovascular Research 55: 838-849, 2002).

Stock solutions of BH4 (in 20 mM HCl), BH2 and B (in DMSO) were made toa final concentration of 10 mM and stored at −80° C. Calibrationstandard working solutions were prepared from stock solution at 100, 10,7.5, 5, 2.5, and 1 nM in K2 EDTA human plasma modified by 0.1% (w/v)1,4-Dithioerythritol (DTE). Quality control working solutions of BH4,BH2 and B were prepared at 5, 8, 25 and 50 nM in K2 EDTA human plasmamodified by 0.1% (w/v) DTE and stored at −80° C. For sample processing,plasma was diluted 1:10 in resuspension buffer. To 180 μl of the dilutedplasma, 20 μl of the 10× precipitation buffer was added. This process ofplasma dilution and precipitation was applied to all plasma standards,plasma samples and plasma QCs. After the addition of the 10×precipitation buffer, the sample was centrifuged at maximum speed at 4°C. for 5 min to remove non-specific plasma debris. 150 mL supernatantwas then be transferred to a sample vial and then placed on anautosampler for a 100 mL injection.

The mobile phase (2L) was prepared with 13.6 g sodium acetate (50 mM),2.1 g citric acid (5 mM) 36 mg EDTA (48 mM), 49.4 mg DTE (160 mM), and2% methanol by volume in water. The pH was adjusted to 5.22.Resuspension buffer (20 mL) was made with 20 mL of PBS pH 7.4 (50 mM),20 uL of 1 M DTE (1 mM), and 100 mL of 100 uM EDTA. The 10×precipitation buffer (25 mL) was made fresh with 2.88 mL phosphoric acid(1M), 9.39 g trichloroacetic acid (2 M) and 20 mL 1M DTE (1 mM).

Tetrahydrobiopterin (BH4), dihydrobiopterin (BH2), and Biopterin (B)were separated using reverse phase HPLC separation. BH4 was measuredusing electrochemical detection in which BH4 is oxidized by electrode 1to quinonoid dihydrobiopterin (qBH2) and then reduced back to BH4 atelectrode 2. The detector then uses the current generated by thisreduction reaction to determine the concentration of BH4. BH2 and B canbe measured in the same injection using fluorescence detection. Postcolumn oxidation of BH2 using a conditioning guard cell at the optimumpotential, oxidizes BH2 to Biopterin.

HPLC separation was carried out on an ACE C-18 (250 mm×4.6 mm) column, 5μM, at a flow rate of 1.3 mL/min with a run time of 13 minutes.Electrochemical detection settings were E1: +100 mV (background current+500 nA to +600 nA) and E2: −300 mV (background current −50 nA to −60nA). Post-column oxidation was set at 900 mV. Fluorescence detectionsettings were excitation wavelength: 350 nM and emission wavelength: 450nM.

Linearity and range of the method were assessed based on the precisionand accuracy of the standards in plasma and buffer. The standard curveconcentration was established using at least 4-6 non-zero concentrationsfor each analyte. The concentration of the standards was 1, 2.5, 5, 7.5,10, and 100 nM. The results showed a linear fit from 1 to 100 nM forBH4, BH2, and B with R2 of >0.99.

Accuracy was determined by replicate analysis of quality control samplescontaining known amounts (2, 8, 25, and 50 nM) of the analyte andexpressed as a percent accuracy. Precision is also calculated based onthe data from the quality controls. Intra-assay precision andinter-assay precision were evaluated based on the CV %. On threeseparate experimental runs concentrations of each analyte were preparedin plasma and analyzed. In addition 10 nM of BH4, BH2, and B was“spiked” into human plasma samples to determine the accuracy andrecovery. The measurements of BH4, BH2, and B at 8, 25, and 50 nM provedaccurate within 112%-89% and demonstrated precision (CV %) of 2.5%-20%.Spike recovery experiments using 10 nM BH4, BH2, and B in clinicalsamples of human plasma demonstrated recoveries between 70%-130%. Theresults demonstrate that the method is accurate and precise for sampleswith concentrations greater than 2 nM.

To check for the presence of endogenous interference in six differentlots of plasma, 10 nM BH4, BH2, and B were spiked into six differentlots of plasma and the determine accuracy and precision were determinedfor each plasma sample. Selectivity experiments show that the sixindividuals had endogenous baseline BH4 levels of between belowquantifiable limit to 2.48 nM. Similarly, BH2 and B concentrationsranged from 0.02 to 10 nM. The recovery of the 10 nM spiked analytesranged from 69%-87%. The variability (CV %) across the individual plasmasamples and analytes when spiked at 10 nM ranged from 23%-37%. Thevariability of the endogenous levels of BH4, BH2 and B ranged from0-9.96 nM. Together, the results indicate a trend suggesting matrixinterference or loss during extraction, but do not indicate strongselectivity between individuals.

To measure matrix effect standard curves prepared in plasma or bufferwere compared for accuracy (recovery), linearity and correlation.Comparison of the standards prepared in plasma versus standards preparedin buffer demonstrates a modest matrix effect and generally goodcorrelation. All three analytes had excellent linear fits for plasma andbuffer. BH4 and B did not demonstrate significant matrix effects acrossthe concentration range. However, BH2 had less recovery at the higheststandard concentration (100 nM). The quality control samples prepared inbuffer and plasma demonstrated good accuracy. Overall, matrix effectsseem minimal, with a trend toward less recovery in buffer as compared toplasma. Because BH4 and BH2 are readily oxidized, collected plasma andsample buffers should contain anti-oxidants and have low pH whenpossible.

To test the ability to accurately dilute a plasma and buffer samplespiked with 250 nM of BH4, BH2 and B, plasma was diluted using blankplasma in a 3-fold dilution series. The diluted samples were analyzedand compared to the nominal value after the dilution factor was applied.The dilution of high concentrations of BH4, BH2, and B can be accuratelymade. For BH4 the observed concentrations following dilution werebetween 83%-104% accurate for concentrations between 83.33 nM and 3.07nM. BH2 was 74%-80% accurate across the quantitative range (83 nM-3 nM).B was 119%-113% accurate across the quantitative range (83 nM-3 nM).Therefore, a sample that is above the quantitative limit can be dilutedaccurately.

Four concentrations of analytes (2, 8, 25 and 50 nM) were prepared inplasma and frozen for a minimum of 24 hours for one cycle and a minimumof 12 hours for other cycles for a minimum of three cycles. Samples werethawed unassisted at room temperature in between frozen periods. Theaccuracy and variability after each and all free-thaw cycles wasassessed to establish the maximal number of cycles a sample couldundergo. The BH4-, BH2-, and B-containing samples can undergo up to 3freeze-thaw cycles without significant change in accuracy or precisionof the measurement. Plasma samples with 8 nM-50 nM BH4 are 121%-91%accurate and CV % less than 10%. Similarly BH2 measurements were 77%-88%accurate across the quantitative range of the assay. B measurements were98%-99% accurate across the quantitative range with precision (CV %) of5%-8%. The 2 nM sample of BH4, BH2, and B did not prove accurate orprecise following repeated freeze-thaw. Therefore, standards, qualitycontrols and study samples may be frozen and thawed up to 3 times.

Because the analytes are sensitive to oxidation we examined long-termfrozen stability to mimic expected storage conditions. Fourconcentration levels (2, 8, 50, and 100 nM) of BH4, BH2, and B wereprepared in plasma and stored at −70° C. for 8 weeks. Stability sampleswere assayed fresh and at weeks 3, 5, 6, and 8. BH4 and B had good longterm frozen stability. BH2 demonstrated reduced sample concentrationafter prolonged storage. Over the 8 weeks of storage, plasma sampleswith BH4 were 93%-94% accurate and had CV % between 31%-0.21%, with themost variation seen at the 2 nM concentration. BH2 measurements were63%-85% accurate across the concentrations tested with reduced accuracyat the 2 nM and 100 nM concentrations. The precision (CV %) ranged from37% to 18% for these samples. B measurements were 88%-101% accurateacross the concentrations tested with precision (CV %) of 23%-0.14%,with the highest variability at the 2 nM concentration. Together, thisdata supports the recommendation to store samples for up to 8 weekswithout appreciable loss of analyte concentration. BH2 seems to be themost susceptible to degradation (oxidation).

To measure the stability of BH4, BH2, and B in the autosampler, 8 nM ofeach analyte in reconstitution solvent stayed on the autosampler for0.25, 4, and 11 hours. The accuracy and precision of the measurementswere compared. The observed BH4 measurement was accurate within 5% oftheoretical at each time point with accuracy and precision across allthree measurements of 102% and 0.054% respectively. The measurement ofBH2 had decreasing accuracy and increasing variability after 4 hours.After 11 hours on the autosampler about 50% of the BH2 was measured.This indicates poor autosampler stability in run buffer. The measurementof B remained accurate within 125% of theoretical after 11 hours.Therefore, run times of no more than 4 hours are recommended.

To determine injection carry-over, an extracted baseline plasma samplewas inserted after the highest standard concentration 100 nM. This wasdone to mimic the possibility of overestimating the concentration ofanalyte in a low concentration sample due to carry-over. The injectioncarryover of BH4, BH2, and B is minimal and does not account for morethan 1% of the peak area of the 100 nM upper limit of quantitation. Theinjection carryover accounts for approximately 5%-20% of the lower limitof quantitation, based on the average peak area obtained from the lowquality control (2 nM). Therefore, preferably the samples should beordered from lowest to high (i.e., pre-dose first, followed by post-dosesamples) and additional washes to clean the column periodically during arun preferably will be made to minimize potential carryover.

A qualified method which was robust, specific, accurate and precise wasdeveloped. This method is appropriate to quantify the levels of BH4, BH2and B in plasma for pharmacokinetic and drug studies.

All patents, publications and references cited herein are hereby fullyincorporated by reference. In case of conflict between the presentdisclosure and incorporated patents, publications and references, thepresent disclosure will control.

1. A method of orally administering tetrahydrobiopterin (BH4),comprising administering to a human in need thereof a therapeuticallyeffective amount of BH4 or a pharmaceutically acceptable salt thereof,and informing said human that absorption of said BH4 or pharmaceuticallyacceptable salt thereof is increased when it is ingested with foodcompared to when ingested without food. 2.-12. (canceled)
 13. A methodof increasing gut retention time of tetrahydrobiopterin (BH4) or apharmaceutically acceptable salt thereof in a subject comprisingadministering a formulation to said subject, said formulation comprising(1) BH4 or pharmaceutically acceptable salt thereof and (2) an agentthat slows gut motility, wherein the BH4 or pharmaceutically acceptablesalt thereof administered via the formulation has a longer gut retentiontime in comparison with a control formulation of BH4 or pharmaceuticallyacceptable salt thereof not having an agent that slows gut motility.14.-19. (canceled)
 20. An oral formulation of tetrahydrobiopterin (BH4)or a pharmaceutically acceptable salt thereof, comprising BH4 orpharmaceutically acceptable salt thereof and an agent that slows gutmotility. 21.-25. (canceled)
 26. A liquid formulation oftetrahydrobiopterin (BH4) or a pharmaceutically acceptable salt thereof,comprising an aqueous solution of BH4 or pharmaceutically acceptablesalt thereof, an antioxidant, and a pH buffer.
 27. A dry powderformulation of tetrahydrobiopterin (BH4) or a pharmaceuticallyacceptable salt thereof for constitution into an aqueous solution,comprising a dry powder mixture of BH4 or pharmaceutically acceptablesalt thereof, an antioxidant, and a pH buffer. 28.-37. (canceled)
 38. Amethod of making a liquid formulation of tetrahydrobiopterin (BH4) or apharmaceutically acceptable salt thereof, comprising providing anaqueous solution containing BH4 or pharmaceutically acceptable saltthereof; adding an antioxidant and a pH buffer to the solutioncontaining BH4 or pharmaceutically acceptable salt thereof; sparging theaqueous solution containing BH4 or pharmaceutically acceptable saltthereof, before or after addition of antioxidant and pH buffer, with aninert gas or carbon dioxide; and sealing the sparged solution containingBH4 or pharmaceutically acceptable salt thereof, antioxidant, and pHbuffer in a container. 39.-44. (canceled)
 45. A method of separatingdihydrobiopterin and biopterin, or analogs thereof, from a mixturecontaining both base and dihydro forms, comprising: performing reversephase HPLC using a mobile phase comprising an aqueous solutioncomprising methanol, sodium acetate, citric acid, EDTA, and1,4-dithioerythritol, on a mixture containing dihydrobiopterin andbiopterin, or an analog of dihydrobiopterin and an analog of biopterin.46.-48. (canceled)
 49. A method of measuring biopterins using reversephase HPLC coupled with tandem mass spectrometry (LC/MS/MS), comprising:subjecting samples of blood, plasma, tissue homogenates, or urinecomprising biopterins to oxidation; subjecting the oxidized samples toiodometry; passing the oxidized samples through an ion exchange column;measuring total and oxidized biopterins in the samples using HPLC andtandem mass spectrometry; and calculating the amount of reducedbiopterin as the difference between the total biopterins less theoxidized form. 50.-51. (canceled)
 52. A method of quantitatingbiopterins in a mixture of biopterin species, comprising: providing amixture comprising biopterin and at least one of dihydrobiopterin andtetrahydrobiopterin, or analogs of biopterin and at least one ofdihydrobiopterin and tetrahydrobiopterin; separating the biopterinspecies in the mixture by reverse phase HPLC; and in the case oftetrahydrobiopterin and analogs thereof, performing electrochemicaldetection by oxidizing the tetrahydrobiopterin and analogs thereofpresent by a first electrode to quinonoid dihydrobiopterin forms,followed by reducing the quinonoid forms back to tetrahydrobiopterin andanalogs thereof present at a second electrode, and measuring currentgenerated by the reduction reaction to determine the concentration ofspecies; and/or in the case of dihydrobiopterin, analogs thereof,biopterin, or analogs thereof, measuring such species by fluorescencedetection following post-column oxidation of dihydrobiopterin species tobiopterin.
 53. (canceled)